Pigment-dispersing factor

Transcriptional Regulation

One of the most interesting questions in circadian biology is how a molecular cycle is translated into time of day information for the behaving organism. pdf expression is regulated by the Drosophila clock and requires cycling vrille expression. pdf encodes a neuropeptide expressed in the axons of the pacemaker cells, and these projections connect the LNs with target cells in the dorsal brain. PDF protein has been shown to accumulate in the LN axons with a circadian rhythm. The period of this rhythm is shortened by the per^S mutation, and continuous accumulation of PDF in the dorsal brain is associated with arrhythmia and a variety of period changes in adult locomotor activity. PDF mRNA levels do not cycle in wild-type flies. Since continuous expression of vri suppresses PDF protein accumulation without affecting accumulation of pdf mRNA, cycling Vri expression in wild-type Drosophila is likely to contribute to the observed cycling of Pdf protein. Vri may affect Pdf levels by specifying rhythmic expression of a factor involved in translation, maturation, stabilization, transport, or release of the neuropeptide (Blau, 1999 and references therein).

All of these observations point to a likely role for Pdf in coupling a molecular clock to timed behavior; this study has demonstrated that vri conveys essential regulatory signals from the clock to Pdf. There is also evidence that Pdf can in turn influence function of the clock. In the cockroach, microinjection of PDF produces time-dependent shifts in the phase of the locomotor activity rhythm (Petri, 1997). The magnitude of these phase shifts (up to 4 hr) is similar to that produced by light (Petri, 1997). This indicates that a transient change in Pdf level will cause a stable change in molecular components of a clock that regulates behavior in at least some insects. Possibly, the novel pathway of per and tim suppression observed in V2 and V3 Drosophila lines is a direct consequence of eliminating Pdf (Blau, 1999).

Regulation of the Drosophila pigment-dispersing factor (pdf) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors Clock and Cycle severely diminish pdf RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (s-LN_vs). This clock-gene regulation of specific cells does not operate through an E-box found within pdf regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the s-LN_vs. This posttranslational rhythm is eliminated by period or timeless null mutations, which do not affect PDF staining in cell bodies or pdf mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate pdf or its product in novel and different ways. Coupled with contemporary results that show a pdf-null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks (Park, 2000).

To assess the effects of clock mutations on pdf expression, the normal cellular distribution of the Drosophila gene's native products were examined. By in situ hybridization, the expression pattern of pdf mRNA has been shown to be similar to that determined with anti-crab-PDH. There are four positive cells in each larval brain hemisphere; these persist into adulthood and become the small ventrolateral neurons (s-LN_vs), whose neurites project into a dorsal region of the adult brain. Four large ventrolateral neurons (l-LN_vs) also express pdf; these emerge during metamorphosis and send projections into the optic lobe and across the brain midline. Larvae and adults also contain pdf mRNA in the posterior extremity of the CNS (Park, 2000).

Northern blots reveal no daily rhythm of pdf mRNA abundance, but they could have failed to detect pdf mRNA cycling in a subset of the cells. Thus temporal in situ hybridizations were performed; neither category of pdf-expressing neurons exhibit systematic fluctuations in signal intensities. Therefore, there is no pdf mRNA rhythm for clock mutations to affect (Park, 2000).

Anti-Drosophila PDF antibodies give cell labeling identical to that obtained by in situ hybridization. Neither method leads to marking of cells in the dorsal brain of adults that are stained by anti-crab-PDH. This indicates that the dorsally located antigen is cross-reacting material and does not have to be considered in terms of effects of clock mutations on pdf expression (Park, 2000).

Expression of pdf in the arrhythmic Clk^Jrk mutant has been found to be strikingly abnormal. In Clk^Jrk brains, neither pdf mRNA nor PDF is detectable in larval LN cells and in the s-LN_vs of adults. The same defects were observed in mutant animals heterozygous for Clk^Jrk and a deletion of the locus. These results suggest that Clk is required for pdf transcription, although only in certain cells: the larval LNs and the s-LN_vs into which they develop. Dorsally projecting axonal processes arising from the s-LN_v cells terminate near the calyx of the dorsal-brain mushroom body. In accord with the absence of perikaryal s-LN_v immunoreactivity, these projections are absent from Clk^Jrk brains. In contrast, expression in the l-LN_vs and abdominal-ganglionic cells of adults is apparently unaffected by Clk^Jrk and D); this includes normal staining of centrifugal and interhemispheric projections within the fly's head. However, certain features of projections from l-LN_v cells are aberrant in Clk^Jrk. Approximately 50% of the mutant brains showed abnormal projections; in others, one or two axons from this region project further and irregularly toward a dorsal or median region of the brain. None of these projections is similar to the more dorsal-reaching projections in the brains of wild-type adults (Park, 2000).

Because the Cyc protein cooperates with Clk in their transcriptional-activation roles, pdf expression was examined in cycle mutants. The effects were similar to but less severe than those caused by Clk^Jrk. Most of the larval LNs homozygous for either of two cyc⁰ mutations show much weaker expression of both mRNA and peptide, as compared with wild type, but the mutant expression levels are variable even within a single brain hemisphere: some cells contained signal, whereas others are extremely difficult to detect. The numbers of antibody-stained s-LN_vs in cyc⁰ adults are well above zero, compared with the elimination of such signals in Clk^Jrk flies. Numbers of l-LN_v cells in the brains of cyc-mutated adults are normal, similar to the results obtained in the Clk^Jrk background. About 25% of the adult cyc⁰ brains exhibit an abnormal dorsal projection. In approximately 30% of these mutant specimens, the projections are asymmetric within a single individual: one hemisphere can contain a bundle of dorsally projected axons; but in the contralateral hemisphere, only one or two axons project into the dorsal brain. In other cyc⁰ adults, axons project irregularly into a median brain region (Park, 2000).

The major conclusions from examining pdf expression in the Clock and cycle mutants are that (1) both genes appear to be positive regulators of pdf RNA levels but only in the s-LN_vs and their larval precursors; (2) the effects of Clk^Jrk are stronger than those of the cyc⁰ mutations; and (3) there are developmental defects, because PDF-containing processes in the adult CNS are aberrant in both types of mutants (Park, 2000).

Do Clk and Cyc activate pdf transcription directly? If that is the case, there could be an E-box in this gene's regulatory region. Indeed, within a 2.4-kb segment 5' to the pdf ORF a CACGTG sequence ~1.4 kb upstream of the transcription-start site has been found. The 2.4-kb DNA fragment was fused to the (yeast) GAL4 gene; transgenic strains were generated and crossed to flies carrying UAS-lacZ. The doubly transgenic progeny show faithful beta-galactosidase-reported expression of pdf. To determine whether the E-box is important for the pdf's transcriptional activation, further transgenics were generated. Deletions missing either half or all of the E-box are sufficient to drive brain expression indistinguishable from that observed in wild type. Interestingly, the smallest 5'-flanking region examined mediates the normal brain pattern but does not lead to abdominal-ganglionic expression in the larval CNS. That the influences on pdf expression of Clock and cycle do not operate through a circadian E-box, and thus seem to be indirect, is consistent with the lack of pdf mRNA cycling and Clock/cycle-independent expression in the l-LN_v cells (Park, 2000).

No effect of a period-null mutation on pdf mRNA levels had been detectable in previous Northern blottings. Neither per⁰¹ nor a timeless-null mutation affects the RNA's abundance, by Northern blottings and by in situ hybridizations. To search further for regulation by per or tim, adult brains were stained with anti-PDH at different times of day and night. Strikingly, nerve terminals in a dorsal region of the central brain exhibit rhythms of anti-PDH-mediated staining. The neurites that terminate in this region project from the s-LN_v cells. In an LD cycle, the peak and trough times for the nerve-terminal cycling are 1 h after lights-on and lights-off, respectively. Staining levels in the perikarya of s-LN_vs exhibit some fluctuations but no regular pattern. The adult-specific, larger PDF neurons also exhibit no appreciable cycling of anti-PDH-mediated staining, either in l-LN_v cell bodies or in the termini of their neurites that ramify over the surface of the medulla optic lobe (Park, 2000).

The dorsal-brain, nerve-terminal cycling persists in constant darkness with an ~24-h period in wild type. In that condition the cycle duration is shortened to ~20 h by the per^S mutation, which causes behavioral periodicities to be about 5 h shorter than normal. In the dorsal brains of the per⁰¹ null mutant, nerve-terminal cycling is abolished, and the signal strengths are very low. However, the immunohistochemical procedure performed on these brain sections is not very sensitive. Therefore, a quantitative fluorescence method was used, the better to judge PDF staining intensities in whole-mounted brains. At the peak and trough time-points, nerve-terminal signals in wild type are again higher in the early morning compared with the early night. This temporal difference is not observed in the dorsal brains of the arrhythmic per⁰¹ and tim⁰¹ mutants. In per⁰¹, the staining intensities at both times are nearly identical and at levels intermediate between the per⁺ peaks and troughs. In tim⁰¹, the PDF terminal signals are also the same at the two time-points but significantly higher than in tim⁺ and. The mutational effects of these clock genes on daily fluctuations of PDF abundance at certain nerve terminals indicate that an aspect of this peptide's regulation is, in one way clock controlled, and in another was posttranslationally regulated (Park, 2000).

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a 'clock' situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (Per) protein, including the ventral lateral neurons (LN_vs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LN_ds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest-activity pattern remains unknown. Targeted expression of Per was used to restore the clock function of specific subsets of lateral neurons in arrhythmic per⁰ mutant flies. Per expression restricted to the LN_vs only restores the morning activity, whereas expression of PER in both the LN_vs and LN_ds also restores the evening activity. This provides the first neuronal bases for 'morning' and 'evening' oscillators in the Drosophila brain. Furthermore, the LN_vs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system (Grima, 2004).

PDF function in resetting signal between Drosophila pacemakers synchronizes morning and evening activity

Daily rhythms of physiology and behaviour are precisely timed by an endogenous circadian clock. These include separate bouts of morning and evening activity, characteristic of Drosophila melanogaster and many other taxa, including mammals. Whereas multiple oscillators have long been proposed to orchestrate such complex behavioural programs, their nature and interplay have remained elusive. By using cell-specific ablation, it has been shown that the timing of morning and evening activity in Drosophila derives from two distinct groups of circadian neurons: morning activity from the ventral lateral neurons that express the neuropeptide PDF, and evening activity from another group of cells, including the dorsal lateral neurons. Although the two oscillators can function autonomously, cell-specific rescue experiments with circadian clock mutants indicate that they are functionally coupled (Stoleru, 2004).

The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LN_v cells; M-cells), whereas another group of clock neurons controls evening activity (CRY⁺, PDF^- cells; E-cells). Transgenic mosaic animals were generated with different circadian periods in morning and evening cells. This study shows by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity (Stoleru, 2005).

Overexpression of the Tim kinase Shaggy (Sgg; Drosophila GSK3) shortens the period by 3-4 h. Sgg expression was driven in all clock cells by crossing tim-GAL4 with flies carrying an EP element inserted at the Sgg locus (EP1576, referred to as UAS-Sgg). The locomotor activity rhythm of tim-GAL4/UAS-Sgg (timSgg) flies in constant darkness (DD) confirmed previous results, in that the period was about 3 h shorter than that of control flies (Stoleru, 2005).

Sgg was expressed exclusively in LN_v cells by constructing a Pdf-GAL4/UAS-Sgg genotype. The Pdf-GAL4 driver is well characterized and drives gene expression only in two clock-cell groups: the PDF⁺ small LN_v (s-LN_v) cells (that is, M-cells) and the PDF⁺ large LN_v (l-LN_v) cells. The driver is inactive in the CRY⁺PDF^- evening cells. Pdf-GAL4/UAS-Sgg (PdfSgg) flies also manifested a short period. The period shortening was less than that of timSgg flies, probably because of weaker expression from Pdf-GAL4 driver in LN_v cells. Sgg expression from an even weaker driver, cry₁₃-GAL4, did not affect behavioural period (Stoleru, 2005).

A close inspection of the behavioural actograms revealed that the period of evening activity is significantly shorter in PdfSgg flies (with a daily advance of about 2 h). This indicates that the pace of E-cells was accelerated, although the period manipulation was restricted to M-cells. An advanced evening peak, without an increase in E-cell Sgg expression, indicates that the faster M oscillator might be setting the E-cell pace. It is therefore proposed that the PDF⁺ cells influence molecular circadian events within E-cells (Stoleru, 2005).

To investigate this possibility, the molecular period (cycle duration) of each clock-cell group was estimated in these different genotypes: UAS-Sgg (control), timSgg and PdfSgg. Fly brains were analysed by in situ hybridization for tim RNA expression pattern after 4 days in DD, so that a barely detectable daily advance by 2-3 h would result in an aggregate advance of 8-12 h on DD4 (fourth day of DD). Indeed, Sgg overexpression in all clock neurons (timSgg) markedly shifted the interval of high tim mRNA expression on DD4 by about 12 h, from between CT10 and CT18 to before CT6. (CT is the circadian time within a constant-darkness experiment; CT0 is the hour of the last lights-on event.) All neurons expressing clock genes showed a similar temporal pattern, consistent with the expected Sgg-induced period shortening in all clock cells, and with a deterministic relationship between the molecular period and the locomotor activity period (Stoleru, 2005).

However, the PdfSgg tim RNA profiles were strikingly different and unexpected. Whereas the s-LN_v cells showed a roughly 8 h advance in DD4, expected from a period shortening of 2 h per day, the l-LN_v cells showed no appreciable change from those in control flies; that is, their molecular program is apparently unaffected by Sgg overexpression within these cells. Also surprising were the DN1 and DN3 profiles, which showed a roughly 8 h advance, as were the LN_d cells, which were advanced by about 6 h relative to those in control flies. Since PdfSgg flies do not overexpress Sgg in these three cell groups, their molecular programs behave in a non-cell-autonomous manner. Because the E-cells are included within these groups and because the s-LN_v cells (the M-cells) are the only cells with a cell-autonomous program that match the behavioural period of the flies, the M-cells apparently determine the clock pace of these other neuronal groups, including the E-cells (Stoleru, 2005).

The l-LN_v cells and DN2 cells emerged as the only clock-gene-expressing neurons that evaded control of the M-cells and maintained a wild-type-like phase of tim RNA cycling in PdfSgg flies. Because DN2 cells are genotypically wild type in these flies, it is inferred that they oscillate with cell-autonomous properties and are the best candidates for determining the non-cell-autonomous wild-type-like characteristics of the l-LN_v cells. As a consequence there are at least two parallel clock-cell circuits in the Drosophila brain in constant darkness: the M-E circuit controls locomotor activity rhythms and is driven by the M-cells (s-LN_v cells), whereas the DN2-l-LN_v circuit has as yet unknown functions and is driven by the DN2 cells (Stoleru, 2005).

To verify and extend these concepts, a genotype was generated in which the E-cells should run faster than M-cells. By adding the previously described Pdf-GAL80 repressor construct to the tim-GAL4;UAS-Sgg background, Sgg was expected to be overexpressed in all clock neurons with the exception of PDF-expressing cells. As these include the M-cells (s-LN_v cells), they should run more slowly (24 h) than the E-cells (about 21 h). A 'faster takes all' rule predicts that the short-period E-cells will dominate over the normal 24 h M-cells in this genotype and generate a behavioural rhythm of about 21 h. Alternatively, dominant M-cells will give rise to a behavioural period of 24 h despite the faster endogenous oscillator in the E-cells (Stoleru, 2005).

Consistent with a dominant M-cell model was the observation that timSgg/PdfGAL80 flies had an almost wild-type period in DD. The molecular analysis is also consistent, since the s-LN_v cells manifested a wild-type-like program: tim mRNA peaked between CT12 and CT20 on DD4. Despite Sgg overexpression, the LN_d cells, DN1 cells and DN3 cells had a similar and wild-type-like pattern of tim expression. As described above, this indicates that all three cell groups behave non-autonomously and are probably driven by the s-LN_v cells. This result is supported by the anatomical pattern of s-LN_v neuronal processes, which project towards the brain regions populated by LN_d, DN1 and DN3 cells. DN2 cells were again the only Sgg-overexpressing cells in which the phase of tim RNA oscillation corresponded to the predicted accelerated pace. The l-LN_v cells, despite lacking Sgg overexpression (because of the PdfGAL80 transgene), also showed a comparable advance of tim expression. These timSgg/PdfGAL80 results confirm that the s-LN_v cells determine the phase of LN_d, DN1 and DN3 cells and that an independent cellular network includes the DN2 and l-LN_v cells. Because the behavioural period was wild-type-like and paralleled the molecular clock within the s-LN_v cells, the results confirm that these M-cells assign the circadian period in the absence of light cues (Stoleru, 2005).

To confirm the lack of a contribution of DN2/l-LN_v to the E-M network function and to locomotor rhythms, the timSgg/cryGAL80 genotype was also examined. It is similar to the timSgg/PdfGAL80 genotype described above, except that Sgg overexpression is repressed in a wider group of cells. These include most if not all of the E-cells and l-LN_v cells as well as the M-cells. Since DN2 cells are the only clock cells in which cry promoter-driven expression was not detected, it is expected that the faster clock in timSgg/cryGAL80 would be limited to CRY^- cells, including the apparently cell-autonomous DN2 cells (Stoleru, 2005).

Indeed, tim hybridization in situ confirmed that the period of DN2 rhythm was shortened by about 2-3 h per day. The l-LN_v neurons were shifted to about the same extent, which is consistent with the notion that they behave non-cell-autonomously and follow the pace of the DN2 clock program. All other clock cells maintained a pattern similar to that of control flies. Because timSgg/cryGAL80 flies had a normal behavioural period, these results confirm that l-LN_v and DN2 cells do not contribute detectably to locomotor activity rhythms. This conclusion is in agreement with previous results showing that wild-type flies have persistent DD behavioural rhythms, despite protein oscillation idiosyncrasies of the l-LN_v and DN2 cells (Stoleru, 2005).

How does the M-cell (s-LN_v) clock determine the period of E-cells (LN_d cells/DN cells)? Although previous work indicated possible oscillator coupling and a direct effect of LN_v on the transcriptional oscillations of other clock cells, it was difficult to envision how the M-cells could override the intrinsic molecular timing of the E-cells. A second possibility is therefore considered, namely that the E-cells maintain an unaltered intrinsic clock program but receive a daily resetting signal from the M-cells. This model predicts that the timing of the evening activity within every cycle (between two consecutive mornings) reflects the status of the endogenous clock of E-cells, whereas the overall period exhibited by the evening peaks reflects the pace of the M-cell resetting signal (Stoleru, 2005).

To examine this possibility, the different transgenic strains were assayed for their average evening activity phase within a cycle, by using the leading morning peak as a reference and then measuring the average time until the subsequent evening peak. The overall DD period correlated with the genotype of M-cells as expected, but the length of the subjective day (M-E interval) correlated only with the genotype of the E-cells. In control flies with a period of about 24 h, the subjective day was roughly 12 h, similar to the duration of subjective day of PdfSgg. The latter strain features a wild-type-like E-oscillator but a fast, Sgg-expressing M-oscillator and a period of about 22 h. In contrast, timSgg flies express Sgg in both E-cells and M-cells, and both the average length of subjective day and the period (M-M) are reduced. The results indicate that the E-cells run an autonomous clock program whose starting (or ending) points are determined by daily resetting signals from the M-cells (Stoleru, 2005).

A DD unidirectional M ---> E resetting mechanism also predicts that a slower (24 h) M-cell clock and a faster E-cell clock will have a normal morning peak phase but an advanced evening peak phase. To test this prediction, the behavioural outputs of timSgg/PdfGAL80 and timSgg/cryGAL80 flies, which differ only in the genotypes of their E-cells, were compared. Both strains have periods of about 24 h, but the former should give rise to a fast E-cell molecular program, whereas the latter should have an E-clock of 24 h as a result of suppression of Sgg expression (Stoleru, 2005).

Indeed, the evening phase of timSgg/cryGAL80 is similar to that of control flies, and it always occurs about 2.5 h later than that of timSgg/PdfGAL80. The evening phase of timSgg/PdfGAL80 is more similar to that of timSgg, although the latter genotype has a much shorter period than the former. The length of subjective day of timSgg/PdfGAL80 flies further confirms that the evening phase within each cycle is a reflection of the endogenous E-cell rhythm, whereas the period of the cycle (M-M) correlates with the intrinsic M-cell clock (Stoleru, 2005).

These comparisons indicate that the circadian network is modulated by intercellular communication signals, which achieve and maintain circadian coherence -- the proper relationship between morning and evening activity. The dominant M-clock determines the period of the entire system by providing a daily reset signal to the E-clock in darkness and is therefore a true cellular Zeitgeber. Because the M-cells can delay as well as advance E-cells, the resetting signal may be required for E-cell oscillations. The usual candidate for this signal is the M-cell-specific neuropeptide PDF. It contributes to the normal synchrony and/or rhythmicity in constant darkness, with a striking similarity to the mammalian neuropeptide VIP. Moreover, injecting PDF into the cockroach brain causes circadian phase delays. Other principles and/or molecules may also be relevant to the M-E subnetwork, because E-cells can drive clockless M-cells to manifest cyclical behavioural outputs under 12 h light/12 h dark (LD) conditions (Stoleru, 2005).

The l-LN_v and DN2 cells are the two neuronal groups that escape the M-cell reset signal in DD. They constitute a second circadian subnetwork with no apparent effect on locomotor activity rhythms and no known function. The DN2 cells are among the few clock-gene-expressing brain cells in larvae and are also the only clock cells that do not change their morphology after eclosion. Larval DN2 cells are apparently devoid of CRY and manifest anti-phase oscillations of Tim and PER. It is therefore likely that both the DN2 cells and the l-LN_v cells impart circadian regulation to unknown physiological functions relevant to both larvae and adults. More generally, it is expected that the organizational principles of the two subnetworks described in this study will also be relevant to mammalian neuronal networks with important behavioural functions, for example the relationship between different oscillators in the SCN (Stoleru, 2005).

Electrical hyperexcitation of lateral ventral pacemaker neurons desynchronizes downstream circadian oscillators in the fly circadian circuit and induces multiple behavioral periods

Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LN_V) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LN_Vs synchronize oscillations in downstream clock neurons was tested by rendering the LN_Vs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LN_Vs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LN_Vs synchronize multiple oscillators in the fly circadian pacemaker circuit (Nitabach, 2006).

Understanding the mechanisms for synchronizing multiple independent neural oscillators in circadian circuits is a key issue in circadian biology. This study provides evidence that the excitability state of the LN_V subset of clock neurons plays a critical role in coordinating multiple oscillators in the fly brain. When the LN_Vs are made electrically hyperexcitable by genetically targeted expression of a voltage-gated sodium channel cloned from a halophilic bacterium, NaChBac, transgenic flies exhibit complex free-running behavioral rhythms with multiple periods along with desynchronization of clock protein cycling throughout the pacemaker circuit and disrupted cycling of PDF levels in the dorsomedial terminal projections of the small LN_Vs (sLN_Vs) (Nitabach, 2006).

Anti-PDF immunofluorescence was observed in the dorsomedial terminals of the sLN_Vs in control flies. However, anti-PDF immunofluorescence in the dorsomedial terminals of the sLN_Vs of experimental flies expressing NaChBac in the LN_Vs is maintained at constitutively higher levels. This result is unexpected if PDF release at nerve terminals is the only cellular function influenced by alterations in cellular electrical excitability. Although there remains a formal possibility that NaChBac expression does not cause increased electrical excitability in pacemaker neurons, this is considered highly unlikely because of the robust and opposite effects of NaChBac expression compared with open-rectifier potassium-channel expression on behavior, reciprocal rescue of behavior by coexpression, clock oscillation, and direct electrophysiological recordings of muscle and photoreceptor neurons expressing NaChBac. Furthermore, hyperpolarization of LN_v membrane potential after the targeted expression of open-rectifier potassium channels to these cells causes accumulations of PDF in the cell bodies of the LN_Vs, providing further evidence that membrane potential regulates the rates of synthesis and/or trafficking of PDF as well as release. These results together suggest that regulated electrical excitability of the sLN_V plasma membrane underlies cycling PDF levels in the dorsomedial terminals, and that rendering the sLN_Vs hyperexcitable through NaChBac expression disrupts one or more of the cellular processes (synthesis, trafficking, or release) that determine PDF accumulation in the dorsomedial terminals. It remains unclear whether changes in neuronal membrane excitability directly influences PDF accumulation or whether this is caused by indirect effects via the molecular clock, because PDF accumulation appears to be restricted to pacemaker neurons (Nitabach, 2006).

The behavioral and circuit alterations caused by NaChBac expression in the LN_Vs may be attributable in part to an altered pattern of PDF release or a yet-unidentified neurotransmitter released by the LN_Vs, or to complex circuit properties of the pacemaker circuit. Regulated membrane electrical excitability of other neuropeptide-secreting neurons of the insect nervous system is known to be essential for appropriate control of the temporal patterns of peptide release. PDF may act as an intrinsic coupling signal within the circadian clock circuit that synchronizes multiple oscillators that otherwise free-run independently. This interpretation is consistent with a synchronizing role for PDF proposed on the basis of gradual phase dispersal within the sLN_V subgroup of Pdf⁰¹-null mutant flies in constant darkness. In addition, the results are consistent with the idea that temporally regulated PDF release by the LN_Vs synchronizes the circuit, and are inconsistent with the hypothesis that PDF plays a purely permissive role (Nitabach, 2006).

Recent electrophysiological evidence in another insect suggests a mechanism for PDF- and GABA-mediated synchronization of multiple oscillators of pacemaker circuits (Schneider, 2005). Extracellular multiunit recordings of the candidate circadian neurons in excised preparations of the cockroach accessory medulla exhibit ultradian oscillatory action potential firing that is synchronized by local application of pressure ejected PDF and GABA through glass micropipettes or bath applied GABA (Schneider, 2005). Similarly, circadian neurons in the fly may fire in PDF-regulated assemblies. Although there is as yet insufficient electrophysiological evidence to allow direct comparison of the results in Drosophila with this recent finding in the cockroach, this raises the interesting possibility that NaChBac expression in the Drosophila LN_Vs may result in desynchronized firing of pacemaker neurons throughout the circuit, starting with the LN_Vs themselves. This would be consistent with the biophysical property of NaChBac of low-threshold voltage activation. Interestingly, similar mechanisms for oscillator coupling at the circuit level may also be important in mammals. GABA also modulates phase coupling between the ventral and dorsal oscillators in brain slices prepared from the rat SCN (Nitabach, 2006).

The behavioral results confirm that the Drosophila circadian control circuit contains multiple clocks capable of oscillating independently and capable of independently controlling the pattern, but not the amount, of locomotor activity. They further indicate that properly regulated electrical excitability of the LN_Vs (and perhaps of particular importance, the LN_Vs) is required to synchronize these multiple clocks throughout the pacemaker neuronal circuit. The synchronization of multiple oscillators appears to be necessary to generate coherent single-period behavioral rhythms (Nitabach, 2006).

The reciprocal suppression by NaChBac of the arrhythmicity induced by Kir2.1, and by Kir2.1 of the complex rhythmicity induced by NaChBac, strongly supports the interpretation that NaChBac and Kir2.1 have opposite effects on the electrical excitability of the LN_Vs, with Kir2.1 decreasing excitability and NaChBac increasing excitability. When expressed individually in the LN_Vs, K⁺ channels and Na⁺ channels have opposite behavioral effects: hyperpolarizing K⁺-channel expression results in arrhythmic behavior, whereas depolarizing Na⁺-channel expression results in hyper-rhythmic behavior. The coexpression of these two channel types together results in functional reciprocal compensation, yielding nearly normal behavior (Nitabach, 2006).

In a previous studies, LN_V membrane potential was manipulated to be hypoexcitable through the targeted expression of modified open-rectifier or inward-rectifier potassium channels (Nitabach, 2002). This caused behavioral arrhythmicity and cell autonomous dampening of the free-running molecular clock in LN_V neurons in constant darkness, along with no discernable changes in the cycling of the molecular clock in downstream pacemaker neuronal subgroups at circadian day 2. Those results are consistent with the findings that desynchrony of downstream cell groups does not become apparent in pdf⁰¹-null mutant flies until 2 d in constant darkness. In the present study, LN_V hyperexcitability induces trans-synaptic changes in the free-running temporal pattern of clock protein accumulation in the dorsal neuron subgroups DN1 and DN2. Thus, the DN neuronal groups appear to be functionally downstream of the LN_V neurons in the pacemaker circuit. In negative control flies, the DN1s oscillate in phase with the sLN_Vs and LN_Ds, maintaining synchrony on both days 2 and 5 after release into constant darkness from a diurnal 12 h light/dark entraining regime, whereas the DN2s gradually advance from synchrony in 12 h light/dark to a 12 h phase difference by circadian day 5. The DN2s of control flies exhibit peak PDP1 accumulation at CT14 on day 2 in constant darkness and at CT10-CT14 on day 5 in constant darkness. This gradual shift of DN2 PDP1 oscillation from synchrony with the other cell groups in LD to a 12 h phase advance after 5 d in constant darkness is consistent with observations of DN2 PER cycling. In pdf>NaChBac1 flies expressing NaChBac in the LN_Vs, the DN1s exhibit a PDP1 molecular peak 8 h earlier than control flies on day 2 in constant darkness, and by circadian day 5 this peak has significantly damped and an additional significant peak has appeared at CT22. The DN2s of pdf>NaChBac1 flies exhibit a peak of PDP1 accumulation at CT14 on day 2 in constant darkness, in phase with control flies; by day 5 in constant darkness they peak at CT6, 4–8 h earlier than in controls. This phase shift suggests that the DN2 molecular oscillator of pdf>NaChBac1 flies is running faster than that of control flies. These differences in the temporal pattern of PDP1 accumulation in the DN1s and DN2s induced by NaChBac expression in the LN_Vs indicate that properly regulated electrical activity is required for normal patterns of molecular oscillation in these dorsal cell groups (Nitabach, 2006).

The DN2s may be capable of independently driving behavioral outputs, and are possibly the cellular substrate for the ~22 h short-period component of the complex behavioral rhythmicity exhibited by flies expressing NaChBac in the LN_Vs. The cellular substrates for the ~25.5 h long-period component are likely to reside in other cells within the circuit. In control pdf>TM3 flies, robust free-running PER oscillation is observed in the sLN_V,LN_D, and DN1 neurons after 5 d in constant darkness, with trough levels of PER in the second half of subjective day. The differences in the spatiotemporal pattern of PER accumulation induced by NaCh-Bac expression in the LN_Vs confirm, as indicated by the effects on PDP1 accumulation, that hyperexcitation of electrical activity in the LN_Vs causes desynchronization of the coupling and phase of molecular oscillation in dorsal clock neurons (Nitabach, 2006).

Multiple oscillators are distributed throughout the pacemaker circuit in Drosophila. The present study confirms and extends evidence for multiple oscillators in the pacemaker circuit in Drosophila. The independent oscillators driving the multiple period components of the behavioral rhythms that were observed do not appear to correspond directly to the 'morning' and 'evening' oscillators, which have been localized to the LN_Vs and LN_Ds, respectively. The current results emphasize that the activity of the LN_Vs controls the synchronization of independent oscillators throughout the pacemaker circuit. The normal pattern of DN1 and DN2 clock oscillation requires properly regulated electrical excitability of the LN_Vs. Further, the results suggest that the DN2s, and at least some other cell groups, possess independent output pathways to the downstream locomotor circuitry (Nitabach, 2006).

This study introduces a novel method for inducing electrical hyperexcitability in neurons of interest by the expression of the low-threshold voltage-gated sodium channel NaChBac. This method is likely to be useful for the analysis of other neural circuits. In another study (Luan, 2006), the utility of the NaChBac channel for enhancing excitability in other neurons has also been demonstrated. Targeted expression of ion channel subunits in vivo provides a powerful means for precisely perturbing neuronal membrane excitability to probe the role of activity on neuronal development and function. Initial methods to exogenously regulate electrical excitability in neurons in vivo have used potassium channel expression to electrically silence neurons. Exogenous manipulation of electrical excitability within specific Drosophila neurons can be combined with finer parsing of neural circuits using GAL80 and other genetic approaches (Nitabach, 2006).

This study has shown that aberrations of electrical excitability in Drosophila neurons, either hyperexcitability induced by NaChBac or hypoexcitability induced by Kir2.1, can be rescued by coexpression of an ion channel with an opposite effect on excitability. This provides reason to believe that such an approach to neurological disorders of aberrant electrical activity such as epilepsy might indeed be feasible (Nitabach, 2006).

Protein Interactions

PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors

The neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. A Class II (secretin-related) G protein-coupled receptor (GPCR) has been identified that is specifically responsive to PDF and also to calcitonin-like peptides and to PACAP. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems (Mertens, 2005).

An antiserum against the final 20 amino acids of the predicted C terminus of PDFR was used to establish sites of PDFR expression within the adult brain. In the wild-type adult brain, the most prominent PDFR immunosignals revealed a large cell body in the dorsal-lateral protocerebrum. Roughly 20 neuronal cell bodies were stained in the anterior and medial subesophageal ganglion (SEG). In addition, scores of more weakly stained soma were detected in all regions of the brain, especially along the superficial aspects of the medulla. Strong staining of neuronal processes was evident throughout the central brain and optic lobes. These immunosignals were lost when the antibody was preincubated with the immunizing peptide, but were not altered in either the pdfr P1 or P2-36 mutant stocks (Mertens, 2005).

Throughout the brain, PDF-positive processes were always associated with PDFR-positive processes. For example, the projection of the small LNv neurons was closely associated with PDFR-positive puncta in the dorsal protocerebral neuropil. Single optical sections revealed that the PDF-positive terminals were closely apposed to PDFR-labeled processes. PDF-positive projections within the median bundle were likewise in proximity to abundant PDFR-positive processes. The large PDF-expressing LNv neurons make a broad tangential projection along the distal medulla. Notably, numerous PDFR-stained cells and processes were evident in areas of the medulla and lobula that lacked PDF-stained processes. Along the lateral aspect of the medulla, little evidence was seen of receptor processes immediately adjacent to the tangential PDF projection except in the anterior aspect (Mertens, 2005).

It was asked whether any circadian pacemaker neurons (as assayed through Per immunostaining) coexpressed PDFR. Receptor expression was found in only a subset of defined circadian pacemaker neurons. A prominent pair of DN1 neurons was positive for both Per and for PDFR; these two cells were closely abutted to the dorsal surface of the brain and placed anterior to the other DN1s. Two to three DN3 neurons were weakly PDFR immunopositive. The DN2 neurons, large LNv neurons, LNd neurons and small LNv neurons all lacked PDFR immunosignals. PDFR immunosignals did not vary diurnally. pdfr mRNA did not exhibit diurnal or circadian variation according to results of RNA profiling; instead, it was regulated at a steady-state level by per (Mertens, 2005).

Evidence indicates that in Drosophila PDF signals via a Class II GPCR that is most closely related to the calcitonin-CGRP receptor family. The EC_[50] of ~25 nM measured in this study is likely an overestimate that reflects the heterologous expression system that was employed. PDFR signaling properties in vitro parallel published accounts of PDF actions in vivo. PDF elicits increases in cAMP in vivo, and the PDFR appears coupled to G_s in HEK293 cells. Also, genetic analysis showed that the NF1 protein operates downstream of PDF to support circadian output (Williams, 2001). Similarly, this study found that cotransfection of dNF1 in HEK293 cells greatly increases the efficacy of PDF in producing high-amplitude signaling through the PDFR. This effect is reminiscent of NF1 coupling another Class II peptide GPCR, the PACAP receptor (PAC 1), to adenylate cyclase (Dasgupta, 2003). While Class II peptide GPCRs typically couple to G_s, many Class II receptors also signal via calcium, including CGRP receptors and VPAC receptors). Similarly, PDFR signaling also increased calcium levels, albeit with a much higher EC_[50] value in comparison with the effect on cAMP levels. In all, these data provide a basis for future evaluation of PDF receptor properties in situ (Mertens, 2005).

While PDF-related peptide and DNA sequences appear to be restricted to invertebrate lineages, its receptor (CG13758) is clearly related to certain mammalian receptors (Brody, 2000). These observations suggest that there is conservation of the PDF signaling pathway between arthropods and chordates. PDFR is a Class II peptide GPCR, and other members of this category (e.g., PACAP and VIP receptors) exert profound influences in the mammalian circadian system. In some respects, the functional roles of PDF in the fly circadian system and VIP in the mouse are parallel. Both peptides are required for the normal display of behavioral rhythms in constant conditions -- producing short period rhythms or arrhythmicity -- and both affect the rhythmicity of cellular pacemaking (VIP). Among the mammalian Class II GPCRs, PDFR is more closely related to calcitonin and calcitonin-gene related peptide (CGRP) receptors than to either PACAP or VIP receptors. CGRP immunosignals and CGRP binding sites have been measured in the SCN. Nevertheless, the functional analogies of PDFR-like signaling to CGRP-R-like signaling may be limited, since receptor component protein (RCP), a protein that modulates CGRP responsiveness in a variety of cell types, does not affect PDFR coupling in the current experiments. It is notable that both Drosophila PDFR and mammalian VPAC receptors respond to PACAP peptides. In fact, PDFR was activated by PDF and PACAP-38, also by the peptides calcitonin, adrenomedullin, and a Drosophila ortholog of calcitonin called DH₃₁. Among these, PDF is clearly the most potent ligand and produces the strongest secondary signals. DH₃₁ activates a separate Drosophila Class II GPCR called CG17415, a receptor that is not sensitive to PDF. It is proposed that the PDF receptor displays partial agonism by diverse ligands, which is a common feature among Class II peptide GPCRs. For example, VPAC receptors demonstrate high-affinity interactions with VIP and PACAP and, to lesser extents, with other naturally occurring peptides such as GRF and secretin. PACAP-38 has several physiological effects in Drosophila tissues. Whether PDFR also represents an endogenous PACAP receptor in vivo is now open to investigation (Mertens, 2005).

To what extent can the properties of PDFR explain the in vivo behavioral signaling controlled by PDF? Four of the five Drosophila Class II GPCRs were tested and it was found that CG13758 alone displays sensitivity to PDF. The one untested Class II GPCR is CG12370, and, on the basis of its strong sequence similarity to CG8422, it likely encodes a CRF receptor-related receptor that is sensitive to the peptide DH₄₄. The genetic analysis to date does not allow exclusion of the contribution of other (potential) PDFRs to the regulation of circadian rhythmic behavior. However, the results clearly indicate that PDFR is primarily responsible for PDF signaling underlying the modulation of the Drosophila geotactic behavioral response. Two results underscore this point. pdfr alleles produced a geotactic phenotype as severe as that displayed by pdf mutant flies. Also, flies transheterozygous for pdf and either of two distinct pdfr mutations displayed a strong mutant phenotype, while individual heterozygotyes were not distinguished from controls. Together, these data clearly link the actions of pdf and pdfr within the same physiological pathway. The simplest hypothesis to explain these results is that PDFR is the primary receptor for PDF in the context of geotactic behavior. Why do certain alleles of pdfr display a strong geotactic phenotype, but not a strong locomotor phenotype? Those results are consistent with published properties of the pdf mutant flies indicating that the geotaxis assay is sensitive to small increments of PDF signaling (Toma, 2002). It is proposed that such sensitivity may underlie the differential effects that was measured with receptor mutants. It follows that definition of the complete locomotor phenotype of pdfr mutant flies awaits recovery of stronger mutant alleles. Indeed, this prediction is fully met by analysis of a naturally-occurring mutation of the pdfr (CG13758) locus that produces a circadian locomotor defect which closely matches that of pdf (Lear, 2005). That independent genetic data strongly supports the contention that CG13758 encodes the principle PDF receptor in Drosophila (Mertens, 2005).

In general, excellent correspondence was found between PDFR-positive processes and PDF-positive processes in diverse brain regions. In the dorsal brain, the trajectory and extent of processes from the small LNv neurons are closely matched by receptor-positive processes. Likewise, receptor processes are closely intermingled with PDF-positive varicosities in the anterior medulla and the median bundle. By contrast, many receptor-positive processes were tens of microns away from the closest PDF-positive processes. These distances do not necessarily preclude physiological interactions between PDF and PDFR, as indicated by studies of 'receptor mismatches'. These and other examples support the concept of volume transmission, which refers to the diffusion of bioactive substances across considerable distances via the extracellular space. Given these antecedents and based on the proximity of receptor immunosignals to PDF signals in several areas, it is proposed that the pattern of PDFR expression is consistent with a role in mediating PDF signaling throughout the brain and optic lobes. An additional and nonexclusive hypothesis is that PDFR displays high-affinity interactions with more than one ligand (analogous to mammalian VPAC receptors), and this possibility is supported by its partial agonism, as was observed in vitro (Mertens, 2005).

PDF is a synchronizing factor that sustains or delays molecular oscillations within pacemaker neurons, including oscillations within the pacemakers that release PDF. However, the degree to which PDF acts directly or indirectly on pacemaker cells remains uncertain. The results of studying PDFR-like immunoreactivity do not support the hypothesis of broad, direct PDF action on pacemaker neurons. Among the ~150 brain pacemakers, PDFR immunosignals are only expressed by a pair of DN1s and more weakly by two to three scattered DN3s. The antibody could detect PDFR in most pacemaker cells when the protein was overexpressed, but not in native tissue. These results argue that, normally, most pacemaker neurons contain very low amounts of the receptor. Hence, it is suggested that the predominant influence of PDF on the synchronization of circadian pacemaker neurons proceeds via indirect neuronal connections. Specifically, these results focus attention on the prominent pair of PDFR-positive DN1 cells as potentially critical relay neurons within the circadian pacemaker network (Mertens, 2005).

In summary, the identification of a PDF receptor provides the basis for addressing PDF functions in a cellular context. Its sites of expression define potential sites of PDF actions. Its signaling properties will illuminate the mechanisms by which PDF modifies geotactic behavior and helps organize daily locomotor rhythms. (Mertens, 2005).

A G protein-coupled receptor, groom-of-PDF, is required for PDF neuron action in circadian behavior

The neuropeptide Pigment-Dispersing Factor (PDF) plays a critical role in mediating circadian control of behavior in Drosophila. Mutants have been discovered in groom-of-PDF (gop) that display phase-advanced evening activity and poor free-running rhythmicity, phenocopying pdf mutants. In gop mutants, a spontaneous retrotransposon disrupts a coding exon of a G protein-coupled receptor, CG13758. Disruption of the receptor is accompanied by phase-advanced oscillations of the core clock protein Period. Moreover, effects on circadian timing induced by perturbation of PDF neurons require gop. Yet PDF oscillations themselves remain robust in gop mutants, suggesting that GOP acts downstream of PDF. gop is expressed most strongly in the dorsal brain in regions that lie in proximity to PDF-containing nerve terminals. Taken together, these studies implicate GOP as a PDF receptor in Drosophila (Lear, 2005).

In light:dark cycles, both pdf and gop mutants display reduced or absent anticipation of the morning “lights on” transition and a phase advance of the evening activity peak. In addition, both mutants display free-running rhythms in constant darkness that decay over time. Both mutants also exhibit phase-advanced molecular rhythms after several days in constant darkness. The striking similarity between gop and pdf mutants strongly suggests that these two genes operate in a discrete pathway. Interestingly, both pdf and gop mutants were discovered as spontaneous mutations (Lear, 2005).

Abundant evidence is available that the gene mutated in the gop mutant is the peptide GPCR CG13758. Complementation testing maps the gop phenotype away from other mutations on its X chromosome and to an area of 3A region containing just six candidate genes including CG13758. CG13758 transcripts are severely disrupted by a retrotransposon insertion in the third exon corresponding to a portion of the N-terminal extracellular domain. None of the mutant cDNAs analyzed would produce a wild-type full-length receptor. These data strongly suggest that the disruption of CG13758 is largely responsible for the gop phenotype (Lear, 2005).

Tenetic and phenotypic analysis suggests that GOP acts downstream of PDF: (1) PDF remains rhythmically expressed in gop mutants; (2) alteration of circadian period through manipulation of clock genes in PDF⁺ neurons is blocked in gop mutants, indicating that gop is required for PDF neuronal effects on circadian phase. Although essential for PDF neuron action, the gop transcript is most strongly expressed in cells thought to be the targets of PDF neurons in the dorsal brain, likely including the pars intercerebralis and perhaps the DN1s. Indeed, changes are observed in Per expression in a subset of DN1s in the dorsal brain. Finally, preliminary data is available that the extracellular domain of GOP can bind PDF using an in vitro assay. Moreover, this binding appears to be specific. It is competed by PDF but not by another neuropeptide, proctolin, and significant binding is not observed with an unrelated peptide. Further studies using broader sets of ligands and quantitative affinity assessments in combination with cell-based signaling studies will be necessary to definitively identify GOP as the PDF receptor. The behavioral phenotype described in this study suggests that GOP may be the sole PDF receptor in Drosophila (Lear, 2005).

The gop expression pattern is also consistent with a role as a PDF receptor. gop expression was noted most prominently in the dorsal brain. There is a wealth of evidence that PDF release into the dorsal brain mediates circadian behavior. This study found gop transcript expression in parts of the dorsal brain in the regions of the LNv terminals, including areas near the pacemaker DN1 neurons as well as the pars intercerebralis. The idea is favored that these sites of receptor expression mediate circadian behavior. While these cells cannot be definitely identified as the DN1 group, it is interesting to note that Per expression in a subset of DN1s is altered in gop mutants, consistent with potential GOP function in these neurons. Nonetheless, cell-specific double-labeling experiments will be necessary to demonstrate clearly gop expression in DN1 pacemaker neurons (Lear, 2005).

These studies also clearly reveal a role for GOP in feedback onto the core oscillator. Previous studies described binding of biotinylated PDF directly on subsets of LNs and DNs. No significant gop expression is observed in the LNs yet molecular oscillations in the LNs are altered. The notion is favored that the effects of gop on LN oscillations are not cell-autonomous, but instead reflect altered network function. Anatomic studies have defined a potentially reciprocal circuit between LNvs and DN1s. Such a circuit may be necessary to reinforce molecular cycling under constant conditions. It is proposed that altered signaling in the DN1s due to loss of gop may modulate feedback onto the LNvs and result in advanced Per cycling in these cells. Consistent with this hypothesis reduced Per staining was observed in a subset of DN1s in gop mutants (Lear, 2005).

CG13758/gop encodes a class B GPCR neuropeptide receptor most closely related to calcitonin receptors (Brody, 2000). Of note, rhythmic calcitonin receptor expression has been observed in the mammalian circadian pacemaker, the suprachiasmatic nucleus. More compelling is the apparent role of peptidergic class B GPCR signaling in both fly and mammalian circadian clocks. Like gop mutants, genetic knockout of the VPAC2 receptor, a class B GPCR that is equally activated by VIP and pituitary adenylate cyclase-activating peptide (PACAP), results in a loss or alteration of behavioral and molecular rhythms (Harmar, 2002). Thus, these studies reveal underlying similarities in clock mechanisms beyond the core transcriptional feedback loops. The identification of a G protein-coupled receptor essential for PDF neuronal action represents an important step toward elucidating the molecular and neural pathways that transform core molecular oscillations into daily behavioral rhythms (Lear, 2005).

Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF

The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, a P insertion mutant was isolated that preferred different temperatures during the day and night. This mutation, which was named han, reduces the transcript level of CG13758. Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants are similar to those of pdf null mutants. It was also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, it is proposed that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons (Hyun, 2005).

PDF is expressed in l-LNvs and s-LNvs and is secreted in the axon termini of these neurons. These neurons have large arborizations and send projections to contralateral and dorsal areas of the brain where LNd, DN1, DN2, and DN3 are present. In contrast, the PDF receptor Han is expressed in 13 pairs of neurons in adult fly brains: four l-LNvs, one LNd, seven DN1s, and one DN3. Therefore, it is reasonable to conclude that the PDF signals produced by l-LNvs and s-LNvs are transmitted to four l-LNvs, one LNd, seven DN1s, and one DN3 through Han receptors. LNv, LNd, DN1, and DN3 neurons play certain roles in the control of circadian rhythmic behaviors and, thus, PDF-Han signaling might also play some roles in coordinating interactions between these groups of clock neurons (Hyun, 2005).

Comparing expression patterns of PDF reveals, interestingly, that Han is expressed only in l-LNv but not in s-LNvs. Both l-LNvs and s-LNvs express PDF. It has been suggested that s-LNv neurons are the most important master neurons of clock neurons in flies because strong oscillations of Per and Tim proteins are continuously sustained over five days in s-LNvs in DD. Although neurite fibers of l-LNv and s-LNv are inter-connected, oscillation of Per and Tim proteins in l-LNv is not obvious a few days after the 'light-off' in contrast to s-LNv neurons. Han-mediated PDF signaling may contribute to the coordinated interaction of l-LNv with s-LNv neurons because Han is expressed in l-LNv neurons. These two groups of clock neurons have been shown to control morning oscillators while LNd neurons control evening oscillators (Stoleru, 2004; Grima, 2004). In both pdf and han mutants, the morning oscillators, as well as the evening oscillators, do not function properly. Flies cannot properly anticipate the time when light will be on or off. This implies that PDF-Han signaling in four l-LNv neurons and one LNd neuron, in which Han is expressed, is necessary for the proper function of the morning and evening oscillators (Hyun, 2005).

Han is homologous to the mammalian CT receptor and the VPAC₂ receptor. The mouse VPAC₂ receptor is known to be important for the maintenance of circadian rhythmic behavior and core clock gene expression in mice (Harmar, 2002). Both the CT receptor and the VPAC₂ receptor are expressed in the mammalian clock center SCN. Therefore, it is possible that a similar mechanism to coordinate interactions between clock neurons via PDF-Han signaling may also be present in the mammalian brain (Hyun, 2005).

Screening mutants showing abnormal thermal preference originally led to the isolation of han. han^X7867 prefers a colder temperature than normal only during the night. However, han³³⁶⁹ and han⁵³⁰⁴, which is null, did not show clear differences in temperature preference during day and night. Instead, they consistently preferred a temperature of 23.5°C, slightly colder than normal. It is not known yet if Han has a role in temperature sensation or thermoregulation in flies. Because the pdf⁰¹ mutant has been reported to show abnormal geotactic behavior (Toma, 2002), whether han mutants exhibit any defects in geotaxis was examined by means of a simple climbing assay. In this assay, no significant geotactic phenotypes were observed in han mutants. This observation does not exclude the possibility that some subtle geotactic phenotypes are present in han mutants, which might be detectable by more elaborate analysis with the sophisticated apparatus described by Toma (2002). Han expression in neurons other than clock neurons such as those in ventral nerve cords in the larval brain suggests that Han may play roles other than regulating circadian rhythmic behaviors. The possible roles of Han in other physiological or behavioral phenomena such as temperature preferences or sensation need to be investigated further in connection with circadian rhythms (Hyun, 2005).

DEVELOPMENTAL BIOLOGY

A single Pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of PDF mRNA are consistently higher in males than in females (Park, 1998). There are no detectable hybridization signals in the head of the arrhythmic disconnected/I> mutant, regardless of sex. This seems to contradict the immunocytochemical studies showing that, despite the absence of PDF immunoreactivity in lateral neurons anterior to the medulla optic lobe, 8-16 PDH-immunoreactive neurons near the mushroom-body calix in the dorsal brain are still intact in this mutant (Helfrich-Foster, 1997 and 1998). Therefore, it is suggested that the vast majority of the PDF mRNA is derived from PDF-containing lateral neurons. Alternatively, the dorsally located neurons may be nonspecifically immunostained due to the use of antibody against crustacean peptide. Clarification of this issue awaits the use of a specific antibody raised against genuine Drosophila PDF (Park, 1998).

Antisera against the crustacean pigment-dispersing hormone (beta-PDH) were used in immunocytochemical preparations to investigate the anatomy of PDH-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and in that of several brain mutants of this species, some of which express altered circadian rhythmicity. In the wild-type and in all rhythmic mutants (small optic lobes, sine oculis, and small optic lobes;sine oculis) double mutants, eight cell bodies at the anterior base of the medulla (PDFMe neurons) exhibit intense PDH-like immunoreactivity. Four of the eight somata are large and four are small. The four large PDFMe neurons have wide tangential arborizations in the medulla and send axons via the posterior optic tract to the contralateral medulla. Fibers from the four small PDFMe neurons ramify in the median protocerebrum, dorsal to the calyces of the mushroom bodies. Their terminals are adjacent to other PDH-immunoreactive somata (PDFCa neurons) which send axons via the median bundle into the tritocerebrum. The results suggest a possible involvement of the PDFMe neurons in the circadian pacemaking system of Drosophila. The location and size of the PDFMe neurons are identical with those of neurons containing the Period protein, which is essential for circadian rhythmicity. Changes in the arborizations of the PDFMe neurons in small optic lobes;sine oculis double mutants are suited to explain the splitting in the locomotor rhythm of these flies. In the arrhythmic mutant, disconnected, the PDFMe neurons are absent. The arrhythmic mutant per0, however, shows normal PDH immunoreactivity and therefore, does not prevent the expression of PDH-like peptides in these neurons (Helfrich-Forster, 1993).

The Period protein (Per) is a essential component of the circadian clock in Drosophila. Although Per-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Some Per plus neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons, and they might interact with Per-containing glial cells (Helfrich-Foster, 1995).

In adults, most of the ventral group of LNs (LN_vs) are immunoreactive to the peptide hormone PDH. This PDH immunoreactivity in the group of smaller LN_vs (small LN_vs) persists from early larval stage through the metamorphosis. In the current study per-beta-gal fusion gene construct called BG expression persists in a similar set of LNs. If these BG-expressing LNs in larvae are PDH-immunoreactive, it would support the idea that LNs in larvae are indeed the precursors of a subset of LN_vs in the adults. Thus, L3 brains of BG were double-labeled for X-gal and anti-PDH at ZT 0. The double-labeling procedure was performed on 22 brains (44 brain hemispheres) and gave reliable results on 41brain hemispheres. The numbers of LNs labeled by either BG expression or PDH presence, or both were counted. In ~80% of the valid brain hemispheres, four or five LNs were positive for either BG or PDH, or both. In most of the samples in which five LNs were positive, all of the LNs were stained for BG, but the number of PDH-stained LNs never exceeded four. These PDH-stained LNs also were stained for BG in all 12such cases. In most of the specimens for which four LNs were revealed, all four of the neurons were indeed double-labeled for PDH and BG. These results indicate that there are five larval LNs, of which four contain Per and PDH, suggesting that these four doubly expressing neurons correspond to the precursor of small LN_vs in adults, whereas one LN contains only Per and had not been identified previously in adults. One further feature of the PDH-related results was that the immunohistochemical signals revealed the projections of the four small LN_vs in the double-labeled preparations. Interestingly, their terminals were always located in close proximity to the cell bodies of dorsal neurons-2^Larval (DN2^Ls) (Kaneko, 1997).

Pigment-dispersing hormones (PDH) are a family of octadecapeptides that have been isolated from several crustacean species. An antiserum against the crustacean PDH was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla, known as the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons). This group of neurons seems to be involved in the control of adult circadian rhythms. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and four neurons with small somata (small PDFMe neurons). Both the small and the large PDFMe neurons are identical to the ventral lateral neurons, a group of neurons containing the Period protein. Both subgroups are usually absent in adults of behaviorally arrhythmic disconnected mutants. The compound eyes of these mutants are usually disconnected from the optic lobes due to a severe defect in optic lobe development. disco mutants, as a result, have either very tiny rudiments of optic lobes if no connections are made at all (unconnected phenotype) or, if some connections are established (connected phenotype), the optic lobes have an almost normal size but are grossly disorganized. disco mutants are behaviorally arrhythmic, and the lateral neurons are generally absent in adults. In the wild type, PDH immunoreactivity is seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons persist unchanged into adulthood, whereas the large ones seem to develop halfway through metamorphosis. In addition to the PDFMe neurons, three other clusters of PDH-immunoreactive neurons stain in the developing nervous systems of Drosophila and are described in detail. Two of them are located in the brain, and the third is located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons are absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults (Helfrich-Forster, 1997).

Mutations at the disconnected (disco) locus of Drosophila disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons (the dorsal and ventral lateral neurons) are usually among the missing cells, and most disco flies are behaviorally arrhythmic. Ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that other cells also contribute to the rhythmic control of locomotor activity (Helfrich-Forster, 1998).

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects had been limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons with a beta-PDH antiserum was performed for the entire nervous systems of Phormia and the fruitfly Drosophila. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neuropils of the brain in both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies are found (only six are found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. Some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hindgut (Nassel, 1993).

Axon caliber in monopolar cells L1 and L2 of the fly's lamina can change dynamically. Swelling by day, L2 exhibits a daily rhythm of size changes apparently mediated by wide-field LBO5HT and PDH cells. L1/L2 axon profiles were measured planimetrically in the housefly, Musca domestica, from 1 microns cross sections. Four hours after injecting 5-HT into the optic lobe, L1's axon swells but L2's does not, whereas PDH enlarges both axons. Similar to 5-HT, histamine (the photoreceptor transmitter) enlarges L1 but not L2, mimicking light exposure, while glutamate GABA and both decrease L1 and L2. 5,7-dihydroxytryptamine decreases L2 and, somewhat, L1, an effect attributable to the loss of LBO5HT neurites. Twenty four hours after cutting LBO5HT and PDH commissural pathways, L1 and L2 both shrink. Apparently, the size of L2 depends on either LBO5HT or sufficient 5-HT, and L1 and L2 have different response ranges to 5-HT. Responses to PDH imply that daytime PDH release drives a circadian rhythm, enlarging L1 and L2 (Pyza, 1996).

Circadian control of eclosion: Interaction between a central and peripheral clock

Drosophila displays overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or 'entrain' to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless. Although a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. The LN clock has been shown to be insufficient to drive eclosion rhythms. The prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, it is demonstrated that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event (Myers, 2003).

Lateral neurons (LNs) are considered the central circadian pacemaker. These LNs are required for rest:activity rhythms and are most likely required for controlling the timing of eclosion (adult emergence from the pupal case). Eclosion is considered to be under the control of the circadian system because its timing is gated such that it is restricted to the hours surrounding dawn each day, even for flies that are developmentally ready hours earlier. Because eclosion occurs once in a single fly's lifetime, the multiple events that occur over several days within a population are considered a rhythm. This gating is absent in flies mutant for the clock gene period (per) or timeless (tim) and is also absent in disconnected flies that lack LNs (Myers, 2003).

Neuronal clocks (including those in the LNs) are sufficient to drive rest:activity rhythms, but perhaps not eclosion rhythms. It was of interest to determine whether these clocks would be sufficient for eclosion gating by using fly lines in which the molecular clock functions in neurons only. The gal4-UAS binary system was used to express Tim only in neurons by using the elav^c155-gal4 driver and a UAS-tim transgene in an arrhythmic tim null background. This manipulation does not rescue eclosion rhythms as it does locomotor rhythms in adults (Myers, 2003).

Another circadian mutant line, one that also displays rhythmic rest:activity behavior, was also arrhythmic for eclosion. This fly line, cry^b, is mutant for a circadian photoreceptor, Cryptochrome (Cry). Cry^b affects the sensitivity of the LN molecular clock to pulses of light but does not affect its endogenous rhythm. Emerging evidence now suggests that Cry is a central clock gene in peripheral clocks. Besides demonstrating that the LN clock is insufficient to drive eclosion rhythms, the eclosion data suggest that Cry may also be required within a relevant peripheral clock mechanism (Myers, 2003).

From these data, it is concluded that a LN molecular clock, which can drive rest:activity rhythms, is not sufficient to restore eclosion rhythms. A peripheral clock, then, is necessary to maintain eclosion rhythms, even in the presence of a functioning LN clock (Myers, 2003).

The prothoracic gland (PG), is part of an endocrine structure known as the ring gland. This structure surrounds the heart just anterior to the cardia and is present during all stages of life except adulthood. The PG secretes ecdysteroids, which when converted to the active form of 20-hydroxyecdysone bind to their nuclear hormone receptor (ecdysone receptor, EcR) and affect gene transcription. These alterations in gene expression cause tissue metamorphosis over the course of development. Levels of ecdysteroids peak at the beginning of larval and pupal stages but, during the two days just prior to eclosion, drop to nearly undetectable levels. In Manduca sexta (tobacco hornworm), this drop in ecdysteroid titer is necessary for eclosion to proceed normally (Myers, 2003).

Previous studies suggest that there is some circadian control over PG function. Notably, ecdysone titers cycle in a circadian fashion in Rhodnius prolixus. In Drosophila, Per is present and oscillates in central brain-PG cultures taken from white prepupae. It is not known, however, whether both Per and Tim oscillate in this tissue under free-running conditions (in constant darkness and temperature) immediately preceding eclosion. Presumably, these conditions should be met before one considers the PG a true clock tissue and an appropriate candidate clock tissue involved in the control of eclosion gating (Myers, 2003).

Clock function was assessed in the PG by quantitating Per and Tim levels over the course of the day in intact pupae. Both Per and Tim levels change over the course of the LD cycle, with a significant difference between the peak (late night) and the trough (late day) values. The peak of Per expression is slightly later when compared to Tim. Both expression profiles, though, match those seen in the LNs. Per and Tim still show significant differences in daily expression in constant darkness (DD), although the difference between the peak and trough values is, as in other fly tissues, smaller. Per expression in DD, although significantly different throughout the course of the day, does not match its LD profile. This effect is similar to the delay in Per degradation in DD versus LD seen in head extracts, although the delay is more pronounced within the PG (Myers, 2003).

It is concluded that there is a molecular clock inside cells of the PG at the time when pupae are developmentally ready for imminent eclosion. Because differences between peak and trough levels of Per are smaller, the profiles of daily Tim expression in DD were used to report PG clock function (or clock synchrony within the population) in subsequent experiments (Myers, 2003).

Does circadian output of the PG clock gate eclosion? Because lesions of the PG are lethal, the necessity of the PG clock was established by assaying eclosion rhythms in fly lines in which genetic manipulation had disrupted the clock inside the PG. To disrupt the molecular clock inside the PG, Tim (UAS-tim^2-1) was expressed at all times of day specifically in PG cells by using the Mai⁶⁰-gal4 driver. Expressing Tim in this manner disrupts eclosion rhythms. Peaks are present in the UAS-tim^2-1; Mai⁶⁰-gal4 eclosion profile, but a rhythm and gating are absent. In this line, adult locomotor behavior remains rhythmic (80.3% of adults had a significant circadian period to their rest:activity behavior in constant light. Consistent with a role for the PG in eclosion gating, it was found that there are no significant differences in the daily expression of Tim in the PG of the arrhythmic cry^b flies (Myers, 2003).

Although the clock inside the LNs is not sufficient for eclosion rhythms, the cells still appear to be required for eclosion gating. It is likely that the LNs could control PG clocks, much like the suprachiasmatic nucleus (SCN) of the hypothalamus is believed to drive peripheral clocks in mammals. Anatomical evidence does suggest that the LN axons (containing pigment-dispersing factor, PDF) indirectly innervate the PG (Myers, 2003).

To determine whether LNs are necessary, tests were carried out for the presence of both eclosion rhythms, and a PG clock in a fly line that lacked LN cells. A fly line was used in which LNs were ablated without lesioning many other neurons. This is a more focused disruption than that caused by the disconnected (disco) mutation. LN cells were ablated by driving a cell death gene, head-involution defective (hid), with pdf-gal4. These pdf-gal4 X UAS-hid flies are also arrhythmic for rest:activity behavior as adults. In the PG of pdf-gal4 × UAS-hid and in disco⁰¹ flies, there is no longer any significant difference in Tim expression over the course of the day. Although eclosion gating in the pdf-gal4 X UAS-hid line appears to be present during the first two days in constant darkness, rhythms do not persist. It is speculated that the flies emerging during the first two days of this assay are gated because their exposure to the entraining LD cycle persists until relatively late in development. This may result in limited and short-lived synchrony through unknown mechanisms. Clearly, though, LNs are required to maintain eclosion rhythms (Myers, 2003).

To determine whether a functioning molecular clock inside the LNs is necessary for their influence on the peripheral clock and on eclosion, the effect of disrupting this clock was examined. Tim (UAS-tim^3-1) was expressed in neurons of wild-type flies at all times of day by using an elav^c155-gal4 driver, all in a wild-type background. This perturbation of Tim expression is sufficient to disrupt locomotor rhythms in the adult fly. Although the molecular clock in the LNs is disrupted, there are still daily changes of Tim in the PG, and eclosion remains rhythmic (Myers, 2003).

It is possible that the role of the LNs is to provide, via PDF, a signal to the PG clock. In the LN axons that project to the dorsal brain, PDF expression cycles, with PDF release believed to occur during subjective night. The rhythmicity of PDF release and eclosion correlate well. For instance, in per⁰ and tim⁰ flies, PDF is no longer released in a rhythmic fashion from these dorsal, LN projections, and eclosion is arrhythmic as well. To determine whether PDF is part of the LN output pathway to the PG or involved in eclosion gating, both PG clock function and eclosion were assayed in flies with no functional PDF protein (pdf⁰¹). These flies are arrhythmic for locomotor behavior as well as for eclosion. In the PG, Tim levels were significantly different over the course of the day in the presence of an LD cycle, but neither Per in LD nor Tim in DD showed significant differences in their daily expression profile. These data indicate that the endogenous clock inside the PG cannot function (or entrain) without PDF in the fly. PDF overexpression and anatomical studies suggest that the LNs are the best candidates for a source of PDF relevant to eclosion behavior and the PG clock. PDF, though, is also expressed in a small subset of neurons in the central brain (LN_vs and two to four tritocerebral cells) and in four to six abdominal cells. In addition, there may be other inputs to the PG. For instance, the Tim cycling seen in LD conditions is most likely due to an acute light response, suggesting the presence of photic input to the PG. Supporting this hypothesis are data from R. proxilus, whose PG clock (its presence is inferred from cyclic release of ecdysteroids in culture) is also directly photosensitive (Myers, 2003 and references therein).

Thus, the PG molecular clock is under control of the central clock. This is unlike other Drosophila peripheral clocks, such as those in the antenna, which can operate autonomously. It is also unexpected when one considers data from studies in which PG clock function is directly or indirectly assayed in culture and determined to be independent of the central brain (or independent of any tetrodotoxin-dependent output from the brain, in the case of Drosophila). The PG clock, in fact, is more similar to peripheral clocks in the mammalian circadian system. In addition, it is important to note that edysteroid synthesis (and presumably PG clock function) in the cockroach Periplaneta americana also depends upon input from the central brain. Perhaps, then, the mechanisms controlling peripheral clock function are not the same in different tissues in the same insect or in the same tissue in different insects (Myers, 2003).

From these data, a model for the circadian gating of eclosion emerges. The LNs secrete PDF into the anterior protocerebrum, where it acts on neurons that innervate the PG. Appropriate regulation of PDF levels is critical. Just as the absence of PDF disrupts eclosion, so can excess levels of it in the dorsal brain. However, the mechanisms underlying this PDF overexpression phenotype are unknown (Myers, 2003).

This study introduces a new set of clock cells necessary for the regulation of eclosion rhythms. It is not known, however, whether the LN and PG clocks together are sufficient to control eclosion gating. The current hypothesis holds that, in the ventral nervous system of Drosophila, cells containing Crustacean Cardioactive Peptide (see Cardioacceleratory peptide) are the most likely sites for control of eclosion gating, as indicated by two lines of evidence. The first is that CCAP, in response to eclosion hormone (EH), can activate ecdysis within minutes in Manduca sexta. However, the circadian gate of eclosion cannot be regulated solely by EH or CCAP because flies without either of these sets of neurons still eclose within a circadian gate. The second line of evidence is that some CCAP cells in Drosophila also express LARK, an RNA binding protein that regulates eclosion rhythms. LARK oscillates in a clock-dependent manner inside these cells. Interestingly, PG cells also contain LARK, although there have been no reports of cycling LARK outside of the CCAP cells. The exact mechanism for how and where development and circadian inputs are coordinated to control eclosion gating are still important and open questions (Myers, 2003 and references therein).

Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. Evidence is provided that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component starts from the E activity bout. However, a short-period component originates not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as 'main' oscillator controlling M and E activity bouts in Drosophila (Rieger, 2006).

Daily biological rhythms are governed by inherent timekeeping mechanisms, called circadian clocks. Such clocks reside in discrete sites of the brain and consist of multiple autonomous single-cell oscillators. Within each neuron, interacting transcriptional and translational molecular feedback loops as well as ionic signaling pathways constitute the oscillatory mechanism of the clock. It is not understood how individual pacemaker neurons interact to drive behavioral rhythmicity. The long-standing model of Pittendrigh and Daan (1976) assumes that the clock consists of two groups of oscillators with different responsiveness to light, one governing the morning (M) and the other the evening (E) activity of the animal. Typical M and E activity bouts are present in animals ranging from insects to mammals and suggest that the two-oscillatory model is generally valid. It has been shown that M and E bouts could be eliminated or reinstated by manipulating different circadian pacemaker neurons in Drosophila. This work has suggested that the ventral (LN_v) and dorsal (LN_d) subsets of the lateral neurons are the neuronal substrates for the M and E oscillators. It is not known whether these two oscillators respond differently to light (Rieger, 2006).

The particular power of the two-oscillator model is that it explains observed adaptations to seasonal changes in day length. The model predicts that the M oscillator will shorten and the E oscillator will lengthen its period when exposed to extended constant light (LL). As a consequence, the M activity occurs earlier and the E activity occurs later in long summer days, helping day-active animals avoid the midday heat. The model also predicts that the M oscillator will free-run with short period and the E oscillator with long period when animals are placed in constant light. However, such internal desynchronization between oscillators does not occur, because high-intensity constant light usually results in arrhythmicity. In Drosophila, the clock protein Timeless (TIM) is permanently degraded during light-induced interaction with Cryptochrome (CRY), leading finally to the arrest of the clock. Without functional CRY, this does not happen. Indeed, internal desynchronization into two free-running components (one with a short period and the other with a long period) has been described for cry^b mutants under constant-light conditions. The present study aims to analyze the molecular state of all clock gene-expressing neurons during behavioral rhythm dissociation to test the Pittendrigh–Daan model and refine the neuronal substrates of the E and M oscillators (Rieger, 2006).

This study supports the notion that the activity rhythm of Drosophila is controlled by at least two sets of neuronal oscillators. Furthermore, the definition of these neuronal substrates of both oscillators were refined more precisely than previously. As proposed by Pittendrigh and Daan (1976), the two oscillators show different responses to light: one is accelerated and the other decelerated by constant light. However, a deviation from the original model was observed. In contrast to previous observations, the current results suggest that the PDF-positive s-LN_v cells control not only the M but also the E activity bout. Therefore, the discussion should perhaps not focus of a 'morning' oscillator but rather of an M–E or 'main' oscillator (to keep the 'M'), for the following reasons. The PDF-positive s-LN_v cells are essential for maintaining activity rhythms after several days under constant conditions, and electrical silencing of the LN_v cells severely impairs free-running rhythms. In the present study, the PDF-positive s-LN_v cells appear to dominate the rhythms in those flies that did couple E and M components after the first crossing-over on day 11 in LL, because such flies free-ran with short period (Rieger, 2006).

The hypothesis that the PDF-positive LN_v cells control not only the M activity but also partly the E activity can also explain other findings. The E activity bout is always the most prominent peak, which persists under constant-dark conditions, whereas the M activity bout is much reduced under such conditions and may even disappear. Thus, mainly the E component constitutes the free-running rhythm, and it seems implausible that the neurons responsible for rhythmicity under these conditions should have no impact on the E component. Indeed, it has been found that the s-LN_v show the most robust cycling after extended time under constant conditions. Furthermore, another study emphasizes the importance of the s-LN_v cells for the timing of activity peaks under constant conditions (Rieger, 2006 and references therein).

Despite their dominance, the PDF-positive s-LN_v cells depend on functional LN_d and DN cells to provoke a normal E activity bout under light-dark conditions. Flies with the clock gene PER present only in PDF-positive LN_v cells have a prominent M activity bout but lack the E activity bout. It is unclear whether this is attributable to the E activity fusing with the M activity or whether the E activity is suppressed, but these findings show that the output from the PDF cells requires PER in the LN_d and DN cells to manifest wild-type activity patterns (Rieger, 2006).

It was found that the PDF-negative 5th s-LN_v cell cycles in-phase with the LN_d cells under LL and thus may contribute to the E component. Notably, the PDF-negative 5th s-LN_v cell shows high-amplitude cycling. Although this is not proof of the involvement of this cell, it suggests that it is an important circadian pacemaker neuron. Little is known about this cell because it could not be distinguished from the other lateral neurons in the former studies in which single-labeled clock protein staining was performed, but the PDF-negative 5th s-LN_v cell is the only clock cell beside the PDF-positive s-LN_v cells that appears to work from the first larval instar onward. Thus, it might have the same strong impact on the activity rhythm that has been revealed for the PDF-positive s-LN_v cells. More work is necessary to reveal the role of the PDF-negative s-LN_v cell in more detail (Rieger, 2006).

Additional studies are also necessary to fully reveal the function of the DN cells. The current results suggest that the DN₁ and the DN₃ cells may contain different subclusters. Indeed, the DN₁ cells develop at different times and appear to have distinct projection patterns. It is very likely that some DN₁ cells contribute to the M oscillator whereas others supply the E oscillator. Again, there are data that support this hypothesis: if the lateral neurons (s-LN_v, l-LN_v, and LN_d) are absent as a result of mutation or genetic ablation but the dorsal neurons (DN₁, DN₂, and DN₃) are left intact, morning and evening activity bouts are still present under LD conditions, although with reduced amplitude and changed phase. The DN₂ cells might play a special role for bimodal activity patterns because, in wild-type flies, they cycle 12 h out-of-phase with the s-LN_v and LN_d cells under DD conditions. The present study indicates that this is not the case in cry^b flies under LL conditions, because the DN₂ cells were in-phase with all other neurons on the first day in LL. The same applies for wild-type flies under LD conditions. It has been shown that the DN₂ are indeed pacemaker neurons that cycle independently of the s-LN_v cells. However, despite their autonomous function, the DN₂ cells did not visibly contribute to the activity patterns of the flies under constant darkness. This suggests a minor role of the DN₂ cells in the control of the activity rhythm, but the possiblity cannot be exclude that the DN₂, together with the other DN groups, may contribute to morning and evening activity bouts under certain conditions (Rieger, 2006).

The blue-light photopigment cryptochrome is regarded as the main photoreceptor of the fruit flies' circadian clock. This study shows that the compound eyes are responsible for period shortening and period lengthening of the molecular oscillations in different subsets of pacemaker neurons (the M and E oscillators) under LL. Their special role may lie in the adaptation of the clock to seasonal changes. This is in line with previous findings showing that the compound eyes are necessary for the adequate timing of M and E activity bouts in long summer days and short winter days. Cryptochrome, conversely, appears to lengthen the period in all clock neurons as can be deduced from the periods of the wild-type flies that showed internal desynchronization under 'moonlight LL.' In such flies, the periods of both components were clearly longer than those of internally desynchronized cry^b flies (Rieger, 2006).

The internal desynchronization of activity into long- and short-period components described in this study is reminiscent of previous results for Drosophila mutants with primarily reduced optic lobes or ectopic expression of PDF. Both of these fly strains have ectopic PDF-containing nerve fibers in the dorsal brain that might lead to elevated and/or nonrhythmic secretion of PDF in this brain area and may disturb normal communication between the pacemaker cells. It is unknown whether such a perturbed communication results in internal desynchronization between the s-LN_v and the 5th s-LN_v and extra LN_d as observed in the present study. Dual-oscillator systems have been also described for mammals, but in no case they could be traced to the level of single neurons. Like the circadian pacemaker center of flies, the mammalian pacemaker center, the suprachiasmatic nucleus (SCN), contains a heterogeneous neuronal population. A recent study has shown that internal desynchronization of motor activity into short and long periods similar to the one shown in this study can be provoked in rats by special light schedules. As in Drosophila, both components reflect the separate activities of two oscillators in anatomically defined subdivisions of the SCN. Furthermore, there is some evidence to suggest that the SCN is composed of two oscillating M and E components. These results underline the universality of dual-oscillator systems (Rieger, 2006).

Other studies strongly implicate the PDF-expressing LN_v and the LN_d cells as the respective neuronal loci for the morning and evening activity bouts. Despite the near 12 h phase difference between the morning and evening locomotor peaks under LD, no obvious molecular phase differences between these pacemakers have been observed that would explain them. Work in mammals suggests that the relationship between molecular phase and locomotion is complex. For example, nocturnal and diurnal rodents show the same phases of PER oscillations. Furthermore, different rat strains that displayed unimodal or multimodal activity patterns, respectively, all exhibited the same unimodal rhythm in melatonin synthesis. Individual Nile grass rats changed their activity patterns from unimodal–diurnal to bimodal–nocturnal after introducing a running wheel. Despite this dramatic effect on the activity patterns, the wheel had little effect on the circadian pacemaker, and the spatial and temporal patterns of c-Fos expression in the SCN remained similar. All of these data indicate that the relationship between molecular and behavioral phase is not straightforward. Clearly, a multitude of phase relationships between the molecular rhythm and behavior are possible. Brain regions outside the pacemaker center may be responsible for these different phases as was shown recently for mammals. It appears that the same is true within the circadian system of the fly. The present data show that, during the internally synchronized state, the trough in PER level of all neurons correlates with the main activity bout (the E peak). No second trough appears to correlate with the M peak. However, a second small peak can be seen at closer inspection of the PDF immunoreactivity in the terminals of the s-LN_v. This suggests that the unimodal rhythm in clock protein cycling might be converted into a bimodal output already within the neurons (Rieger, 2006).

During the state of behavioral desynchronization under LL conditions, an internal desynchronization was observed simultaneously in PER oscillations among subsets of pacemaker neurons. One interpretation of these data is that constant light causes internal desynchronization between these pacemaker neurons that then in turn drive the behavioral outputs. However, it must be acknowledged that this is only a correlation, and, although the hypothesis is favored that the split molecular rhythms are driving the split locomotor rhythms, it is possible that they are merely tracking or entraining to a split rhythm driven by other pacemakers. For example, the split rhythms might be driven by subsets of dorsal neurons. The hypothesis is preferred that the split behavioral rhythms were driven by the desynchronized PDF-positive LN_v and the 5th s-LN_v/extra LN_d cells for two reasons. First, accumulating evidence points to the lateral neurons (LN_v and LN_d cells) as major pacemaker cells, whereas the dorsal neurons (the DN₁, DN₂, and DN₃ cells) are not sufficient for locomotor rhythms under constant darkness. Second, in rodents, a similar behavioral desynchronization was correlated with a dissociation of clock gene expression between ventrolateral and dorsomedial subdivisions of the SCN. The established role of this brain center as the circadian clock has led to the uncontroversial conclusion that the split molecular oscillations drive the split behavioral oscillations. It is suggested that the same phenomenon is occurring in main (i.e., small LN_v cells) and evening (i.e., 5th s-LN_v and extra LN_d cells) neuronal oscillators in Drosophila (Rieger, 2006).

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. Flies overexpressing the pacemaker gene per or the morgue gene, a gene that can protect flies from the disruptive effects of constant light when overexpressed with the tim-GAL4 driver, are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF - the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness - is dispensable for rhythmicity in constant light. Molecular circadian rhythms are found only in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light (Murad, 2007).

Recent studies have shown that two groups of cells control circadian behavior. The PDF-positive LNvs are called morning cells (M cells), and the LNds evening cells (E cells), because they control the anticipatory behavior observed before dawn and dusk, respectively. In addition, the LNvs are the cells maintaining circadian behavior in constant darkness and controlling the phase of most circadian neurons of the brain. In their absence, circadian behavior rhythms are lost after a few days in DD. Surprisingly, the current results show that a functional circadian clock in the LNvs is actually not necessary for long-term behavioral rhythms. In flies overexpressing PER, the LNvs are no longer circadianly functional under constant illumination. No oscillation of the circadian protein PDP1 can be detected, and yet these flies remain rhythmic for at least 7 days. Moreover, limiting per overexpression to circadian neurons that do not express PDF is sufficient to obtain circadian behavioral rhythms under constant environmental conditions (Murad, 2007).

It is thought that the neurons maintaining circadian behavior independently of the LNvs are not the E cells. Indeed, when per is overexpressed, no sign of circadian oscillation is seen in the neurons that are thought to control the evening activity: the LNds. In addition, the PDF-negative LNv that might also contribute to the evening activity did not cycle in LL when morgue was overexpressed. Moreover, flies with per overexpression driven by cry-GAL4 were completely arrhythmic under constant light. cry-GAL4 is one of the critical GAL4 drivers used to define the E cells. Importantly, molecular circadian oscillations were detected in only one group of cells when per was overexpressed: the DN1s. Due to the high number of DN3s, it cannot be ruled out that a few cells in the DN3 groups also oscillate. Interestingly, it has been shown that a subset of DN3 neurons can maintain their own circadian oscillations in DD, in the absence of circadianly functional LNvs. However, these DN3 cells were not able to generate rhythmic behavior in DD. While it is possible that light is a necessary cofactor for these self-sustained DN3s to participate in the control of circadian behavior, the hypothesis is favored that it is the DN1s that maintain circadian rhythmicity in LL. This idea is strongly supported by several additional findings. First, the phase of PDP1 molecular oscillations in the DN1s on the third day of LL fits well with the long period of the circadian behavior observed under these conditions in per-overexpressing flies. Second, the behavioral observations made with morgue overexpression also suggest that the critical cells for rhythmicity are not the LNvs, and PER staining in morgue-overexpressing flies gave an independent confirmation that robust circadian molecular oscillations are restricted to the DN1s in LL. Finally, in LNv-rescued cry^b flies, only the DN1s show robust, coherent circadian rhythms in phase with the behavioral rhythms. Remarkably, the DN1s can maintain circadian behavior in LL even when PDF is absent. This indicates that they can work autonomously of LNv output. Interestingly, not all DN1s do oscillate in LL, only about six or seven cells most likely. This shows that the DN1 group is heterogeneous. This is not surprising, since the different groups of circadian neurons were named based on their location in the brain, not on their function or developmental lineage. There is ample evidence for heterogeneity of morphology, gene expression, and behavior within these different groups of cells, including the DN1s (Murad, 2007).

Thus, a subset of DN1s can control and generate circadian behavioral rhythms. They must therefore play an important role in the circadian neuronal circuits. Since ablation of the M cells and E cells results in flies with no morning and evening activity, and no self-sustained rhythms in DD, this could mean that the DN1s are usually functioning downstream of the M and E cells. This is further supported by the fact that in the absence of the neuropeptide PDF—believed to be the critical synchronizing signal secreted by the M cells—the DN1s cannot maintain their circadian rhythms in the long run in DD. The DN1s can thus probably function as a relay connecting the LNvs with the neurosecretory cells of the pars intercerebralis (PI), believed to play an important role in the control of locomotor behavior. A LNvs-DN1-PI pathway has also been suggested based on the anatomical studies of the projections of the small LNvs and the DN1s. The expression of the receptor for PDF in at least a subset of DN1s also supports the existence of a functional connection between them and the LNvs. The implication of this connection is that, in wild-type flies under LL, the LNvs should constantly send a disruptive signal to the DN1s, presumably the nonoscillating secretion of PDF (Murad, 2007).

This leaves the following question: if the LNvs and rhythmic PDF secretion are normally required for the DN1s to be rhythmic, why are the DN1s able to free themselves from the disruptive effects of constant light, while at the same time becoming independent of the LNvs? The results show that an important mechanism is the inhibition of the CRY-dependent light input pathway. Indeed, morgue-overexpressing flies are defective in the CRY-dependent behavioral responses to short light pulses, and cry loss-of-function mutations also result in rhythms driven by the DN1s. In the case of per overexpression, it is presumed that the TIM role is reduced, since one of its major functions is to protect PER from proteasomal degradation. TIM is the target of CRY; thus its reduced importance would result in DN1s that are less sensitive to the CRY input pathway. In addition, overexpression of Shaggy, which inhibits CRY signaling, also results in LL rhythms driven by dorsal neurons. However, under natural environmental conditions, inhibition of the CRY input pathway is probably not required for the DN1s to participate in the control of circadian rhythms. Indeed, even in the polar regions of the globe that experience constant light conditions during the summer, the elevation of the sun varies during the day, and this should result in variations of temperature sufficient to synchronize the DN1 circadian clock (Murad, 2007).

The mechanism by which the DN1s avoid becoming arrhythmic in LL as a result of the molecular arrhythmicity of the LNvs, which should result in constant PDF secretion, is not clear yet. It is possible that the presence of light inhibits PDF signaling and thus promotes the role of the DN1s. Light input could come from the eyes, ocelli, or from the DN1s themselves. Alternatively, the DN1s could induce rhythmic PDF secretion. The fact that PDF is not required for LL behavioral rhythms does not exclude this possibility, particularly since the robustness of the rhythms is improved by the presence of PDF (Murad, 2007).

Interestingly, per and morgue overexpression results in a very similar long period phenotype under LL, which could suggest that these two molecules coincidentally affect the period length of the circadian molecular pacemaker in the same way. In DD, however, per overexpression does affect behavioral period length, while morgue does not. The long period phenotype observed in LL actually probably reflects the fact that the CRY input pathway is not completely blocked in the DN1s of per- or morgue-overexpressing flies. Indeed, under very low light intensity, wild-type flies exhibit a long period phenotype as well. In addition, morgue overexpression does not completely block the CRY-dependent responses to short light pulses. Finally and most importantly, LNv-rescued cry^b flies (in which the CRY input pathway is completely nonfunctional in the DN1s) have 24 hr period rhythms. The LNv-rescued cry^b flies show nevertheless a higher degree of arrhythmicity than normal cry^b flies or than flies overexpressing morgue or per. This might be due to the desynchronization observed within the DN3 group of circadian neurons. Indeed, the DN3s do not appear to be desynchronized in per- or morgue-overexpressing flies (Murad, 2007).

A previous report had already shown that LNv-rescued cry^b flies are partially rhythmic, and this was interpreted as evidence for a functional role of CRY directly in the LNvs. The new results show that expression of CRY in the LNvs is probably not very important for the response to constant light. The DN1s are the important cells for this response. Does this mean that CRY is not a photoreceptor in the LNvs? It is thought that CRY actually does function as a photoreceptor in the LNvs as well. CRY is expressed in these cells, and LNv-rescued cry^b flies show very significantly rescued responses to short light pulses. Preliminary experiments with morgue overexpression limited to the LNvs confirm a predominant role of these cells for light-pulse responses. Thus, the CRY input pathway might mediate response to short light pulses by its action in the LNvs and constant light responses by its action in the DN1s (Murad, 2007).

In summary, the work underscores the importance of the DN1s in the control of circadian behavior and responses to light. Earlier genetic studies have indicated that the DN1s modulate the sensitivity of the circadian network to light:dark cycles of very low light intensity. The current results significantly extend this observation by showing the profound impact the DN1s have on the response to constant light and by demonstrating that these cells not only modulate circadian light responses but can also become the driving force controlling circadian locomotor behavior, and this in the absence of environmental cues and functional LNvs. This confers upon them a unique status among non-PDF circadian neurons. One of the striking results is that genetically identical flies rely either on the LNvs or the DN1s for the control of their circadian rhythms, depending on the presence or absence of light. Indeed, the LNvs determine period length in these experiments with per overexpression in DD, but in LL the DN1s set the pace. That the presence or the absence of light can so remarkably shift the dominance from one cell group to the other strongly suggests that the relative contributions of the LNvs and DN1s to the control of circadian rhythms change during the course of the year, particularly at high latitude. The DN1s, which interestingly generate evening activity, would play a more prominent role in the control of circadian behavior during the long days of the summer, while the LNvs would be more important when photoperiods are shorter (Murad, 2007).

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (Per)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional Cryptochrome (Cry), as opposed to wild-type flies, are rhythmic in constant light, transgenic flies were analyzed expressing Per or Cry in the LN-MO or LN-EO. Under constant light conditions and reduced Cry function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, this study shows that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. It is therefore proposed that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons (Picot, 2007).

The PDF-expressing LNs and the PDF-negative LNs were previously characterized as morning and evening cells, respectively, in LD conditions. Furthermore, the morning LNs were able to drive robust 24-h rhythms in DD, whereas evening LNs were not. This study shows that in LL, the evening LNs drive robust rhythms when cryptochrome signaling is absent or reduced, whereas the morning cells are not able to do so. Surprisingly, the molecular oscillations of both groups can be uncoupled from behavioral rhythmicity, depending on light conditions. In DD, the two LN groups show autonomous molecular cycling, but there is no behavioral output when the LN-EO is cycling alone. In LL (and reduced Cry signaling), both groups still show autonomous cycling, but there is no behavioral output when the LN-MO is cycling alone. It is therefore concluded that light has opposite effects on the behavioral output of the two LN oscillators, activating it from the evening LNs and inhibiting it from the morning LNs (Picot, 2007).

The opposite effects of light on the behavioral outputs do not appear to be related to entrainment, since Per oscillations in both the PDF-positive and PDF-negative LNs are synchronized to the LD cycles even in the absence of Cry signaling. The inhibiting effect of light on the LN-MO behavioral output is abolished when the visual system is genetically ablated. This suggests that the projections of the visual system photoreceptors convey, not only input information to the PDF cells (light entrainment), but also signals to control their behavioral output (light inhibition). It is tempting to speculate that light exerts both effects through a direct connection of the PDF cells with the visual system. The Hofbauer-Büchner eyelet photoreceptors that project directly to the LN-MO neurons and participate in the entrainment provide a possible pathway (Picot, 2007).

It was recently reported that the overexpression of Per or of the Shaggy (Sgg) kinase in the PDF-negative clock neurons induced rhythmic behavior in LL. The rhythmicity was associated with the cycling of Per subcellular localization in some of the DNs, whereas the PDF-expressing cells were molecularly arrhythmic. These studies therefore concluded that some DN subsets are able to drive behavioral rhythms in LL. Different groups of PDF-negative cells may thus be able to drive behavioral rhythms in constant light, depending on whether and how the molecular clock is manipulated. Such manipulation could also directly affect molecular oscillations, making them less easy to detect. Since Cry does not appear to have a core clock function in the brain, these data are largely based on situations in which the clock mechanism is little if at all altered. The data support a major contribution of the LN-EO to the robust rhythms of cry^b mutants in LL (Picot, 2007).

The strong rhythmicity of the cry^b pdf⁰ double mutants in LL contrasts with their weak rhythmic behavior in DD. Altogether, these results strongly suggest that this robust rhythm is generated by the LN-EO, which would therefore behave as a PDF-independent autonomous oscillator. However, the period of the oscillator is clearly influenced by PDF signaling, and thus by the LN-MO, going from 24–25 h in cry^b to 22–23 h in cry^b pdf⁰ flies. An attractive possibility is that the strong short-period rhythm observed in the cry^b pdf⁰ double mutant in LL has the same neuronal origin as the weak short-period rhythm described for pdf⁰ mutants in DD. The cellular basis of this PDF-independent oscillator in DD remains unclear, although the presence of similar rhythms in flies genetically ablated for the PDF-expressing neurons suggests that it originates from other clock cells (Picot, 2007).

Different results were obtained for the recently described DN-based LL oscillators. When transferred to a pdf⁰ background, all SGG-overexpressing flies were found to be arrhythmic, whereas about 60% of the Per-overexpressing flies displayed long-period rhythms. This suggests that different types of DNs with different sensitivity to PDF may have been analyzed in these two studies. Although some DNs may contribute to the PDF-independent rhythms, the data suggest a strong contribution of PDF-negative LNs to the rhythmic behavior that persists in pdf⁰ mutants. The weakness of the short-period rhythm of pdf⁰ flies in DD may reflect the inhibition of the LN-EO output in the absence of light (Picot, 2007).

These results indicate that whereas the LN-MO autonomously drives rhythmic behavior in constant darkness, the LN-EO plays this role in constant light, if Cry signaling is abolished or reduced. It is thus suggested that in natural LD conditions, Drosophila behavior could be driven by the LN-MO during the night, and by the LN-EO during the day, when cryptochrome is quickly degraded by light. This supports a model of a light-induced switch between the circadian oscillators of the LNs that would allow a better separation of the dawn and dusk activity peaks in day–night conditions. It has been shown that PDF-expressing LNs drive the clock neuronal network in short days, whereas PDF-negative DN subsets take the lead in long days. Thse results suggest that the PDF-negative cells of the LN-EO could also be a major player during the long days. Surprisingly, it was found that light does not seem to act on the molecular oscillations, but inhibits the LN-MO behavioral output and promotes the LN-EO behavioral output, which may provide an efficient fine tuning of the contributions of the two oscillators. It therefore appears that the visual system controls both the input (entrainment) and the behavioral output of the LN oscillators in the Drosophila brain clock. In species such the honeybee or the flour beetle, which appear to lack a light-sensitive Cry protein, this role of the visual system may be particularly important (Picot, 2007).

Pigment dispersing factor-dependent and -independent circadian locomotor behavioral rhythms

Circadian pacemaker circuits consist of ensembles of neurons, each expressing molecular oscillations, but how circuit-wide coordination of multiple oscillators regulates rhythmic physiological and behavioral outputs remains an open question. To investigate the relationship between the pattern of oscillator phase throughout the circadian pacemaker circuit and locomotor activity rhythms in Drosophila, the electrical activity and pigment dispersing factor (PDF) levels of the lateral ventral neurons (LNv) were perturbed, and their combinatorial effect on molecular oscillations was assayed in different parts of the circuit and on locomotor activity behavior. Altered electrical activity of PDF-expressing LNv causes initial behavioral arrhythmicity followed by gradual long-term emergence of two concurrent short- and long-period circadian behavioral activity bouts in ~60% of flies. Initial desynchrony of circuit-wide molecular oscillations is followed by the emergence of a novel pattern of period (PER) synchrony whereby two subgroups of dorsal neurons (DN1 and DN2) exhibit PER oscillation peaks coinciding with two activity bouts, whereas other neuronal subgroups exhibit a single PER peak coinciding with one of the two activity bouts. The emergence of this novel pattern of circuit-wide oscillator synchrony is not accompanied by concurrent change in the electrical activity of the LNv. In PDF-null flies, altered electrical activity of LNv drives a short-period circadian activity bout only, indicating that PDF-independent factors underlie the short-period circadian activity component and that the long-period circadian component is PDF-dependent. Thus, polyrhythmic behavioral patterns in electrically manipulated flies are regulated by circuit-wide coordination of molecular oscillations and electrical activity of LNv via PDF-dependent and -independent factors (Sheeba, 2008).

The period values of the short and long-period activity bouts (~22.5 and ~25 h) observed in these studies closely match those reported earlier for several clock mutants of Drosophila. The fact that robust multiple behavioral rhythms are observed in numerous mutant backgrounds strongly suggests that homeostatic behavioral resynchrony to period values of ~22.5 and ~25 h is a circuit-level constraint rather than a property of any single subgroup of clock neurons. Nevertheless, it is noted that the two periods that emerge in the mutant cry^b under dim LL undergo continuous and opposite changes in period length with increasing light intensity. Thus, it is likely that inputs perceived by individual components of the pacemaker circuit may differentially influence components of the circuit during the intermediary or transition states, whereas the steady state period is determined by the entire network. Although many studies have proposed the dominance of the sLNv in determining the free-running rhythm in DD, previous reports suggest that the molecular oscillations in DN1 neurons play an important role in determining rhythmic activity/rest behavior both under DD and LL conditions. The results of a study which manipulated molecular oscillations in circadian pacemaker circuit by targeted expression of Shaggy (Sgg; a clock component whose overexpression speeds up the oscillations in mRNA of circadian genes) suggests that whereas sLNv, LNd, DN1, and DN3 cells are part of a circuit that regulates locomotor activity rhythm, the lLNv and DN2 cells form a separate and independent circuit that apparently does not influence locomotor activity rhythm and that the DN2 cells are the dominant component of this second circuit and regulate the oscillations in the lLNv (Sheeba, 2008).

Is there a clear association with the phase of molecular oscillation in any of the circadian pacemaker neurons with the phase of behavioral locomotor activity? Under LD, wild-type flies show two peaks in locomotor activity, yet, all known pacemakers exhibit a synchronized phase of Per oscillation that peaks coincident with the morning peak in activity. Wild-type flies when subjected to DD show a single peak in activity (apparently a derivative of the evening peak), and the peak in Per levels of all cells remain synchronized for the first few days, occurring at the trough of behavioral activity. After ~5 d in DD, the phase of peak Per levels appears to drift apart among the members of the circuit, with DN2 cells being out-of-phase, as determined by sampling at 12 h or 6 h intervals. When Per is overexpressed in the pacemaker circuit using the tim-GAL4 driver, it is possible to induce a robust single-period rhythm under LL in >90% of flies. In this case, molecular oscillation appears to persist in DN1 up to the fourth day in LL as quantified by cell counts based on PDP-1 staining. The peak in PDP-1-immunopositive cell numbers coincides with falling levels of locomotor activity (although not the trough). Two simultaneously occurring behavioral rhythms are seen in LL in cry^b mutants, with increasing fraction of flies showing polyrhythmic locomotor behavior with increasing light intensity. Per levels in the PDF positive sLNv and the PDF negative fifth sLNv are antiphasic as determined by sampling at time points (12 h intervals) corresponding to the peak activity of each of the two polyrhythmic bouts in LL in cry^b mutants and a subset of LNd appears to be in phase with the PDF negative fifth sLNv and has high Per levels during one of two activity peaks (the short-period bout apparently derived from the LD morning peak). For flies expressing voltage-gated sodium channel (NaChBac) in the LNv, the phase distribution of Per cycling between the different pacemaker neuronal subgroups shows an antiphasic relationship between the PDF-positive and PDF-negative subsets of sLNv, but is opposite to that seen in cry^b flies under LL (sampled at ~6 h intervals). Furthermore, two peaks in PER levels are seen in DN1 and DN2 and also a clear oscillation in LNd that is not seen in controls or any other study in prolonged DD. Thus, the association between the short-period and long-period activity bouts with the PDF positive and negative sLNv is labile and is highly dependent on environmental or intercellular signals. Furthermore, the polyrhythmicity in behavior and novel pattern of synchrony among the different members of the circadian pacemaker circuit in NaChBac flies appears to be a result of the alteration of electrophysiological properties in either the sLNv, lLNv, or both types of LNv (Sheeba, 2008).

To summarize what has been observed for the temporal relationship between clock cycling in the pacemaker neurons and overt locomotor behavior, from these results and those from previous studies, it is concluded that there is no absolute relationship between activity level and the phase of Per cycling in sLNv. This is particularly clear when comparing the experimental conditions which lead to two behavioral activity peaks, including the morning and evening bouts for wild type flies in LD; and polyrhythmic bouts seen for LL-cry^b; DD-NaChBac expressed in the LNv. Furthermore, it is proposed that the relationship between the pattern of molecular oscillations among the different neuronal subgroups and rhythmic activity rest behavior in the absence of external time cues under DD and LL is likely to be a result of continuous recalibration of signals being perceived by members of the pacemaker circuit. When these signals are below a certain threshold the circuit remains fairly tightly synchronized in terms of the molecular oscillation of clock proteins. Whereas when membrane electrical properties of parts of the circuit are altered to produce higher-than-threshold signals, molecular oscillations in the circuit are rendered asynchronous along with a loss of temporally regulated behavioral activity (Sheeba, 2008).

The circuit gradually recovers from perturbations applied to parts of the network via circuit-wide plasticity as demonstrated by the emergence of novel behavior pattern with novel consolidated activity patterns and synchrony in molecular oscillation in different subgroups of the circuit. The electrically perturbed LNv do not recover their normal pattern of spontaneous action potential firing. Previous work shows that current injection evokes firing in otherwise silent large LNv. The absence of spontaneous firing in this previous study is probably caused by technical differences in the recording procedures. The results of the current study provide empirical evidence for the idea that gradual resynchrony among a large number of constituent oscillators could account for initial arrhythmicity and subsequent emergence of rhythmic behavior seen in animals when exposed to LL. More recent studies in mammals indicate a mechanism for plasticity in neural networks and outputs involving emergence of a coherent oscillation from previously asynchronous oscillator cells in sub areas of SCN when animals are transferred to DD from LL. In summary, the adult circadian pacemaker circuit exhibits electrical-activity dependent circuit-level plasticity of oscillators as shown by the reorganization of molecular oscillation and rhythmic activity/rest behavior. Electrophysiological studies of pacemaker cells in the accessory medulla of cockroaches has led to the hypothesis that pacemaker cells are organized into assemblies with distinct phases which are synchronized by neurotransmitters such as PDF and GABA. In Drosophila this study shows clear evidence for the existence of PDF-independent factors that contribute toward the regulation of circadian activity/rest rhythm also suggesting the existence of compensatory/redundant mechanisms in the pacemaker neuronal circuit. It is hypothesized that the emergence of rhythmic activity due to NaChBac expression using pdfGAL4 driver in pdf⁰¹ flies may cause the rhythmic release of yet unknown neurotransmitters that are otherwise released at lower levels than would be necessary to elicit robust rhythmic behavior. Electrophysiological measurements of NaChBac expressing flies have shown that lLNv show giant action potentials, which may likely trigger downstream neurons to release, signals that can compensate for the lack of PDF. Together, the results of the current experiments along with those of others that have examined the relationship between activity and the pattern of phase distribution among the different pacemaker components suggests that different genetic manipulations and environmental conditions place the architecture of the pacemaker circuit to unique steady states, each of which is able to use different components of the circadian pacemaker circuit to generate distinct circadian patterns in behavior (Sheeba, 2008).

Circadian control of membrane excitability in Drosophila melanogaster lateral ventral clock neurons

Drosophila circadian rhythms are controlled by a neural circuit containing ~150 clock neurons. Although much is known about mechanisms of autonomous cellular oscillation, the connection between cellular oscillation and functional outputs that control physiological and behavioral rhythms is poorly understood. To address this issue, whole-cell patch-clamp recordings were performed on lateral ventral clock neurons (LN_vs), including large (lLN_vs) and small LN_vs (sLN_vs), in situ in adult fly whole-brain explants. Two distinct sizes of action potentials (APs) were found in >50% of lLN_vs that fire APs spontaneously; large APs originate in the ipsilateral optic lobe and small APs in the contralateral. lLN_v resting membrane potential (RMP), spontaneous AP firing rate, and membrane resistance are cyclically regulated as a function of time of day in 12 h light/dark conditions (LD). lLN_v RMP becomes more hyperpolarized as time progresses from dawn to dusk with a concomitant decrease in spontaneous AP firing rate and membrane resistance. From dusk to dawn, lLN_v RMP becomes more depolarized, with spontaneous AP firing rate and membrane resistance remaining stable. In contrast, circadian defective per⁰ null mutant lLN_v membrane excitability is nearly constant in LD. Over 24 h in constant darkness (DD), wild-type lLN_v membrane excitability is not cyclically regulated, although RMP gradually becomes slightly more depolarized. sLN_v RMP is most depolarized around lights-on, with substantial variability centered around lights-off in LD. These results indicate that LN_v membrane excitability encodes time of day via a circadian clock-dependent mechanism, and likely plays a critical role in regulating Drosophila circadian behavior (Cao, 2008).

This study investigated the temporal regulation of membrane excitability of PDF-expressing LN_v clock neurons. RMP and AP firing patterns in lLN_vs were characterized, and it was demonstrated that lLN_v membrane excitability oscillates as a function of time of day in LD. This oscillation requires a functional circadian oscillator, as it is abolished in per⁰ flies. This requirement could be cell autonomous to the lLN_vs, as suggested by the fact that the oscillation is abolished in DD, a condition in which lLN_v, but not other clock neuron, transcriptional oscillation ceases. sLN_v RMP is most depolarized near lights-on (Cao, 2008).

lLN_v spontaneous activity is somewhat variable, with 33% of cells silent in the light phase and 38% in the dark in LD. That, overall, 35% of lLN_vs are silent in LD is slightly higher but still comparable with mammalian clock neurons in the suprachiasmatic nucleus (SCN). Despite the 35% of silent lLN_vs, the average AP firing frequency and the percentage of lLN_vs that fire APs show a circadian temporal pattern similar to that in mammalian SCN, which is consistent with linear regression analysis of the scatter plot of individual cells. Interestingly, in contrast to the cyclic changes in lLN_v RMP, membrane resistance and AP firing rate decrease from dawn to dusk in the day but stay low during the night in LD. The biophysical basis for this uncoupling of RMP from AP firing rate and membrane resistance at night is not clear, but presumably reflects differential modulation of particular ion channel subtypes. This issue bears further investigation in the future using physiological, pharmacological, and genetic techniques (Cao, 2008).

lLN_vs exhibit two sizes of APs, with the small APs approximately half the amplitude of the large APs. Gap junction inhibitors reduce the frequency of small APs and alter the normal firing rate of large APs, suggesting that gap junctions are involved in modulating membrane excitability of lLN_vs. This is consistent with observations of cockroach lateral neurons, where gap junction blockers also influence spontaneous AP firing rate, and clock neurons in mammalian SCN. However, none of the three gap junction blockers had any effect on the amplitude of either large or small APs, thus making it highly unlikely that APs generated in coupled cells are propagating passively to the site of recording at the soma through gap junctions. That cutting the posterior optic tract removes small APs strongly supports that small APs originate in the contralateral optic lobes. This raises the interesting possibility that the ipsilateral and contralateral arbors of individual lLN_vs can process information independently (Cao, 2008).

WT lLN_v membrane excitability is modulated as a function of time of day. In particular, in the light phase of LD, lLN_v RMP becomes more hyperpolarized from dawn to dusk, as the AP firing rate and lLN_v membrane resistance decrease. In the dark phase, lLN_v RMP becomes more depolarized from the beginning to the end of the night, whereas the AP firing rate and membrane resistance stay relatively stable. This continuous cyclical modulation of WT lLN_v RMP is consistent with previous coarse time-scale studies demonstrating that lLN_v RMP is more depolarized in the early morning and more hyperpolarized in the early night (Park, 2006; Sheeba, 2008). This temporal modulation is almost completely abolished in per⁰ flies, thus demonstrating for the first time its dependence on an intact transcriptional feedback oscillator. Although this is consistent with an autonomous requirement for transcriptional feedback oscillation in the lLN_vs themselves, because per⁰ flies lack transcriptional feedback oscillation in all neurons, this could be a circuit-based phenomenon. To further address this question, the modulation of WT lLN_v excitability was examined on the first day in DD conditions (DD-D1), in which transcriptional oscillation in lLN_vs, but not in any other clock neurons, is abolished. The absence of oscillatory modulation of lLN_v excitability in DD-D1 further supports the conclusion that transcription feedback oscillation is required cell-autonomously in the lLN_vs, and the transcriptional feedback oscillator directly modulates lLN_v membrane excitability at the cellular level. The apparent difference between the demonstration of an absence of dependence of electrical excitability on time of day on DD-D1, and Sheeba's (2008) demonstration of presence of a dependence of electrical excitability on time of day on DD-D14 actually makes sense in light of what is known about the response of lLN_vs to constant darkness. Whereas sLN_vs and other clock neurons maintain their cellular rhythmicity immediately after transition from LD to DD conditions, the lLN_vs immediately lose their cellular rhythms with transition into DD, and only regain cellular rhythmicity after several days in DD. This immediate loss of lLN_v cellular rhythmicity followed by resumption several days later perfectly accounts for the fact that no rhythm of lLN_v electrical excitability was observed on DD-D1 whereas Sheeba (2008) observed such a rhythm on DD-D14 (Cao, 2008).

Either pdf gene mutation, LN_v ablation, or LN_v electrical silencing severely disrupts free-running locomotor rhythms, indicating that PDF-expressing LN_vs play a critical role in controlling circadian behavior. Both sLN_vs and lLN_vs express PDF. However, previous studies suggest that of these two cell groups it is the sLN_vs that are most important for controlling free-running rhythms in DD. In particular, restoration of per gene expression in sLN_vs, but not lLN_vs can rescue free-running circadian rhythmicity in per⁰ null mutant flies. Furthermore, as mentioned above, lLN_v transcriptional feedback oscillation cease in DD. The lLN_vs have extensive dendritic arborizations in the medulla and project to contralateral lLN_vs through the posterior optic tracts, thus enabling enable them to potentially receive light information and synchronize the two hemispheres. The lLN_vs also project to the accessory medulla (aMe), where the sLN_vs have short dendrites that may be postsynaptic to lLN_v projections. Although the functional role of lLN_vs in circadian rhythms is mostly unknown, it is reasonable to speculate based on this anatomy that lLN_vs relay environmental light information to the sLN_vs via synapses formed in aMe. It is thus proposed that circadian modulation of lLN_v membrane excitability in LD gates the transfer of entraining light information to the sLN_vs, which more directly control circadian locomotor rhythms (Cao, 2008).

Because sLN_vs are thought to be pacemakers of the neural circuit controlling circadian rhythms, it is critical to understand how membrane excitability of sLN_vs is regulated, and how sLN_v membrane excitability is transformed to signals that control downstream neurons, and, ultimately, circadian locomotor activity. Recording was performed of 103 sLN_vs in LD, and it was found that sLN_v RMP is most depolarized around lights-on, with greater variability around lights-off. The two linear regression fits were selected to span the 6 h just before, and just after, lights-on (ZT0, ZT24). sLN_v RMP exhibits significant trends of depolarization before lights-on and hyperpolarization after lights-on. It is clear that during the 12 h surrounding lights-off (ZT6-ZT18), there is much more variability in sLN_v RMP. This difference makes some sense, as sLN_vs are considered to be the 'morning' cells that drive the increase in locomotor activity that anticipates lights-on. This would be consistent with an increase in sLN_v electrical excitability in the hours preceding lights-on, and a decrease after lights-on, as was clearly observed. Because the sLN_vs are not thought to be important for driving the increase in locomotor activity that anticipates lights-off (ZT12), it is perhaps not surprising that there are no consistent trends in sLN_V electrical excitability surrounding lights-off (ZT12). The reason sLN_vs exhibit little spontaneous activity in this brain explant preparation is not clear, but it may reflect technical issues relating to the extremely small size of their soma. Previous studies indicate that the peak of anti-PDF staining in the sLN_v nerve terminals is around lights-on, and the trough is around lights-off. It is thus possible that the trough of PDF accumulation around lights-off represents depletion of the releasable pool of PDF starting in the early day, when sLN_vs are most depolarized (Cao, 2008).

This study, and that by Sheeba (2008), demonstrate a dependence of lLN_V electrical excitability on time of day in LD and after many days in DD, respectively. The results go beyond those of Sheeba (2008) in a number of important ways, including demonstration that the daily rhythm of lLN_V electrical excitability depends on a functional cellular circadian oscillator, demonstration of a daily rhythm of electrical excitability in the sLN_V pacemaker subset of clock neurons, and elucidation of the cellular origin of the bimodal distribution of sizes of lLN_V APs. Future studies will determine the daily rhythms of electrical excitability of other functionally and anatomically identifiable subsets of fly clock neurons (Cao, 2008).

The GABA_A receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. Sleep-wake modulation was assessed by a core set of circadian pacemaker neurons that express the neuropeptide PDF. It was found that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. RNA interference-mediated knockdown of the GABA_A receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo (Chung, 2009).

It is proposed that GABA release inhibits large LNv output and PDF release to reduce wake, suggesting an important role for GABA inhibition. In this model, the circadian clock times PDF neuron activation and PDF release during the late night and following day to promote waking behavior. Of note, a similar arousal-promoting function for circadian pacemaker neurons has been described in mammals. This is also approximately the time when the large LNv have been shown to be more depolarized and have higher levels of spontaneous activity. RDL receptors on LNv soma and on fibers in the accessory medulla suggest that GABA may regulate LNv excitability. It is interesting that GABA is also an important neurotransmitter in mammalian circadian pacemaker neurons, capable of reducing their spontaneous activity. In addition, RDL receptors on PDF varicosities in the optic lobe may function presynaptically to regulate PDF release. GABA may also act through metabotropic GABA_B receptors, which have been described in the sLNv, but their function in circadian or sleep behavior is unknown. GABAergic signaling may affect the function of the transcription factor ATF2, which is important for PDF neuron function in sleep. Changes in PDF neuron function may in turn act by antagonizing sleep-promoting circuits that exist within the mushroom bodies as well as the pars intercerebralis (PI). Of note, the PI appears to express the PDF receptor. Identifying the anatomic targets of PDF as well as the neural sources of GABAergic inputs will be important for further defining sleep-wake circuits in Drosophila (Chung, 2009).

Comparative analysis of Pdf-mediated circadian behaviors between Drosophila melanogaster and D. virilis

A group of small ventrolateral neurons (s-LN(v)'s) are the principal pacemaker for circadian locomotor rhythmicity of Drosophila melanogaster, and the pigment-dispersing factor (Pdf) neuropeptide plays an essential role as a clock messenger within these neurons. In comparative studies on Pdf-associated circadian rhythms, it was found that daily locomotor activity patterns of D. virilis were significantly different from those of D. melanogaster. Activities of D. virilis adults are mainly restricted to the photophase under light:dark cycles and subsequently became arrhythmic or weakly rhythmic in constant conditions. Such activity patterns resemble those of Pdf⁰¹ mutant of D. melanogaster. Intriguingly, endogenous D. virilis Pdf (DvPdf) expression was not detected in the s-LN(v)-like neurons in the adult brains, implying that the Pdf(01)-like behavioral phenotypes of D. virilis are attributed in part to the lack of DvPdf in the s-LN(v)-like neurons. Heterologous transgenic analysis showed that cis-regulatory elements of the DvPdf transgene are capable of directing their expression in all endogenous Pdf neurons including s-LN(v)'s, as well as in non-Pdf clock neurons (LN(d)'s and fifth s-LN(v)) in a D. melanogaster host. Together these findings suggest a significant difference in the regulatory mechanisms of Pdf transcription between the two species and such a difference is causally associated with species-specific establishment of daily locomotor activity patterns (Bahn, 2009).

This study has characterized circadian locomotor activity rhythms of D. virilis, a species that has radiated from the melanogaster linage ~63 million years ago, approximately the beginning of the Cenozoic era. During this geological period, major paleoclimatic changes are proposed to drive speciation of Drosophila. In addition, studies indicate diversification of native habitats of Drosophila species, as D. virilis is suggested to be indigenous to eastern Asia, whereas D. melanogaster is believed to be African origin. Therefore, it is possible that D. virilis and D. melanogaster may have evolved unique biological clock systems that suit their endemic environment. This is supported by behavioral data that revealed substantially different daily and circadian locomotor activity patterns between D. virilis and D. melanogaster (Bahn, 2009).

The first insight that Pdf might be responsible for behavioral characteristics of D. virilis comes from the uncanny resemblance in locomotor activity patterns between D. virilis and Pdf⁰¹ mutant flies of D. melanogaster. Under LD condition, activities of the Pdf⁰¹ flies are largely restricted to the daytime; such diurnally shifted activity of the Pdf⁰¹ flies is likely to be attributed to both prominently reduced morning anticipatory behavior and the slight phase advance of evening activity peaks to the photophase. Moreover, like D. virilis, Pdf⁰¹ flies are largely arrhythmic or weakly rhythmic in DD condition. Therefore, it is reasonable to suggest that the lack of Pdf expression in the s-LN_v equivalent neurons is intimately associated with behavioral characteristics of D. virilis. These results are also consistent with morning oscillator functions of s-LN_v's, and further support the importance of Pdf's role within the s-LN_v neurons for lights-on anticipatory behavior (Bahn, 2009).

Daily locomotor activity patterns described for the housefly, M. domestica are notably similar to those of D. virilis, as both species display day-phase-restricted activity without lights-on anticipation. IHC using anti-Pdf showed both large and small LN_v-equivalent neuronal groups in the adult brain of M. domestica. In contrast to this result, in situ hybridization revealed MdPdf mRNA expression only in the l-LN_v-like neurons. These results were confirmed independently. A plausible explanation for this discrepancy is that the s-LN_v-like neurons contain materials that cross-react with the anti-Pdf. From these data, it is tempting to propose that lack of Pdf expression in s-LN_v-like neurons is also responsible for diurnally active locomotion displayed by M. domestica (Bahn, 2009).

In the heterologous host, expression of the DvPdf gene is evident in all of DmPdf-positive groups, suggesting that donor DvPdf regulatory elements are capable of interacting with host trans-acting factors to activate its expression in a manner similar to that of DmPdf. Although no useful information about potential elements emerged from simple sequence alignment between 0.5-kb upstream sequence of DmPdf and 1.9-kb of DvPdf, the cis-regulatory element(s) responsible for s-LN_v Pdf expression is likely conserved in both DmPdf and DvPdf. An important question raised from these studies is, then, Why do D. virilis flies lack DvPdf expression particularly in the s-LN_v-like neurons? It could be that D. virilis does not possess the s-LN_v-like neurons. However, this is unlikely, because a fly species (M. domestica), which is even more remotely related to D. melanogaster, contains Pdf-ir s-LN_v-like neurons, although such immunoreactivity likely originated from cross-reactivity, as mentioned earlier. Attempts were made to confirm the presence of s-LN_v's in the D. virilis CNS using anti-Tim, as was done for D. melanogaster. However, no immunosignals were detectable even in the l-LN_v's at two different time points. Although similarity of the Tim between the two species is substantial (76% overall amino acid identity, perhaps diversity between the two proteins does not allow anti-Tim to detect virilis Tim protein (Bahn, 2009).

In D. melanogaster, DmClk and DmCyc proteins are well-defined upstream positive factors responsible for DmPdf expression specifically in the s-LN_v's. This study study shows that these factors are also essential for the DvPdf transgenic expression in the s-LN_v's, LN_d's and fifth s-LN_v, suggesting that DvClk and DvCyc likely act as positive regulators for the DvPdf in the s-LN_v-like neurons in D. virilis brain. Thus the lack of DvPdf expression in the s-LN_v-like neurons might be due to a loss of function of these proteins in D. virilis (Bahn, 2009).

According to the genome database, DvClk gene (Dvir\GJ11427) predicts to encode a protein of 988 amino acids. RT-PCR result suggests that DvClk encodes a 987-amino-acid product and has differences from the Dvir\GJ11427 at three sites. Two of them are within polyglutamate (Q) stretches, missing two Q's at one position and having the addition of one Q at another position. The other one is a homologous substitution of leucine to isoleucine (data not shown). Amino acid composition of the DvClk shows 70% identity to DmClk. For Cyc, sequence of DvCyc deduced from RT-PCR matches perfectly to that from genome database (Dvir\GJ14003), and amino acid residues of the DvCyc share 85% identity with the DmCyc. In other words, no significant mutations were found within the ORFs of both DvClk and DvCyc that might alter their functions. Moreover, robust activity rhythms displayed by D. virilis under LD cycles, in contrast to significantly abnormal LD behavior of D. melanogaster Clk^Jrk and cyc⁰ mutants, suggest that functions of the two clock proteins are unlikely defective in D. virilis (Bahn, 2009).

Absence of s-LN_v-specific DvPdf expression could be accomplished through negative regulation. According to published work, l-LN_v's and LN_d's in D. melanogaster appear to be originated from the common precursor cells; clusters of these cells are mixed without clear anatomical distinction in the ventral region of early pupal brain. As the pupal development progresses, presumptive LN_d's are separated from l-LN_v's, migrate dorsally, and start to develop their characteristic projections. Shortly after this stage, l-LN_v's become Pdf-positive, while LN_d's remain Pdf-negative. However, one exceptional specimen was found in which the migration of the LN_d's is impaired; interestingly, these neurons are Pdf-positive. These findings are interpreted as follows: activation of the DmPdf in both l-LN_v's and LN_d's during pupal development is a default pathway, and then the suppression of the DmPdf is acquired during the maturation of the LN_d neurons, perhaps through the activation of repressors. These studies provide an interesting possibility of the transcriptional suppression of DmPdf in the LN_d's and fifth s-LN_v. This notion is supported by the ectopic DvPdf transgene expression in these neurons, as negative trans-acting factors might be unable to interact with DvPdf's regulatory region due to sequence incompatibility, thus allowing ectopic expression of the DvPdf. As an extrapolation of these results, it would be interesting to investigate whether negative factors suppress DvPdf expression in the s-LN_v-like (and perhaps LN_d- and fifth s-LN_v-like) neurons in D. virilis. Transgenic dissection of the 1.9-kb DvPdf upstream region will help reveal specific cis-acting elements that are necessary for such negative DvPdf regulation (Bahn, 2009).

A role for blind DN2 clock neurons in temperature entrainment of the Drosophila larval brain

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest-activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. This study investigated how the larval clock network responds to light and temperature. Focus was placed on the CRY-negative dorsal neurons (DN2s), in which light-dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. The phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. These results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network (Picot, 2009).

Although the absence of PDF severely affects Drosophila activity rhythms in DD, the exact function of the neuropeptide in the adult clock neuronal network remains unclear. In LD, PDF is required to produce a morning activity peak and to properly phase the evening peak, but not to entrain the brain clock. The behavioral phenotypes of PDF receptor mutants resemble that of the pdf⁰¹ mutant. PDFR is expressed in all clock neurons except the large ventral lateral neurons (l-LNvs), supporting a role of PDF in maintaining phase coherence within the adult clock network in DD. The loss of PER oscillations in the DN2s of pdf⁰¹ larvae demonstrates a clear and novel role of PDF in transmitting not only phase information but also a synchronizing signal without which the receiving neurons are not entrained in LD (Picot, 2009).

The current results show that the CRY-less DN2s are 'blind' neurons that perceive light indirectly. The PDF receptor rescue experiments strongly suggest that PDF acts on its receptor on the larval DN2s themselves, which are located in the vicinity of the LN axon terminals. Furthermore, DN2s possess a wide and dense neuritic network that borders on the axons of the LNs over a large fraction of their length. However, it cannot be ruled out that expression of the receptor in the (PDF-negative) fifth LN is involved in synchronizing the DN2s downstream, through PDF-independent mechanisms (Picot, 2009).

The PDF-negative fifth LN is also a CRY-negative clock neuron, but it cycles in phase with the CRY-positive neurons of the larval brain. The visual input to the PDF-expressing LNs appears sufficient to phase them normally even in cry^b mutants. It could thus be expected to entrain the CRY-less fifth LN in phase with the other larval LNs, as observed, in contrast to the CRY-less DN2s. A direct input from the visual system to the fifth LN is also consistent with its PDF-independent entrainment by LD cycles. Similarly, light entrainment of the larval DN1s in cry^b mutants is consistent with their suggested connection to the visual system. Thus, the CRY-less DN2s would be the only larval clock neurons devoid of such a connection (Picot, 2009).

Adult eclosion rhythms depend on the PDF-expressing LNs and appear to require the PDF-dependent clock that resides in the prothoracic gland. Since the larval DN2s project in the pars intercerebralis, a region of the brain that sends projections to the prothoracic gland, they could play a role in this physiologically important clock function. These results raise the possibility that the damped PER oscillations in the DN2s of the pdf⁰¹ mutants participate to their eclosion phenotype (Picot, 2009).

The DN2s are the only larval clock neurons that are phased identically by light and temperature, but their temperature entrainment appears independent of any LN-derived signal. PER oscillations in the DN2s have a larger amplitude in HC cycles than in LD cycles, also suggesting a prominent role of temperature in their entrainment. Conversely, the molecular oscillations of the PDF-positive LNs have a larger amplitude in LD compared with HC cycles. In the latter, the molecular oscillations of the PDF-expressing LNs seem to follow those in the DN2s, with a large phase change compared with LD conditions. The DN1s and the PDF-negative fifth LN, in contrast, share another phase that is slightly advanced. Interestingly, behavioral and transcriptome data in adult flies indicate that HC cycles result in a general phase advance relative to LD cycles. Cooperative synchronization of the clock by light and temperature likely requires temperature changes to act earlier than light changes since changes in temperature always lag behind changes in solar illumination in nature. The very different relative phasing of the larval clock neurons in HC versus LD cycles suggests different ecological constraints on this life stage, spent mostly burrowed in food, in which light may be a weaker Zeitgeber, and in which the lag between temperature and light changes may be quite different (Picot, 2009).

When a functional clock is absent from the DN2s (and the fifth LN), the larval PDF-expressing LNs are unable to entrain to thermocycles, whereas they autonomously entrain to LD cycles. It remains possible that autonomous temperature entrainment of the larval LNs (but not the DN2s) requires per transcriptional regulation, which the GAL4-UAS system is lacking. But the results demonstrate the existence of a control exerted on the LN clock by CRY-negative clock cells when temperature is the synchronizing cue. Although a role of the fifth LN cannot be ruled out, the absence of autonomous photoperception by the DN2s nicely fits with a role in temperature entrainment. The high cycling amplitude of the DN2s in thermocycles and the locking of the phase of the LNs on that of the DN2s in these conditions strongly support their role in the temperature entrainment of the LNs (Picot, 2009).

Additional studies should investigate whether the DN2s communicate with the LNs via fibers that appear to run along the dorsal projection of the LNs. Alternatively, the dense dendritic-like network of the DN2s could ensure reciprocal exchanges between them and the LNs. A model is thus proposed whereby, in the larval brain, the DN2s and the four PDF-positive LNs form a distinct subnetwork, with the LNs entraining the DN2s in LD, whereas the opposite is true in HC). What becomes of their hierarchy in constant conditions, after entrainment stops? Their relative phases appear to change little at least during the first 2 d after entrainment, whether they have been set in antiphase by LD entrainment, or in phase by HC entrainment. This suggests that, whatever the entraining regimen, the LNs and the DN2s run autonomously in constant conditions. However, it cannot be excluded that one of the two groups still dominates but requires more time after the end of entrainment to shift the phase of the other (Picot, 2009).

The rhythmic behavior of the adult flies that emerge from the temperature-entrained larvae is almost in antiphase compared with the one of flies entrained by light during the larval stage. This strongly suggests that the phase of the adult rhythms is set by the antiphasic oscillations of the larval PDF-positive LNs, consistent with these cells being the only neurons in which molecular cycling persists throughout metamorphosis. It is thus believed that the large phase shift of adult activity can be accounted for simply by the large phase shift of molecular oscillations in the PDF-expressing LNs (Picot, 2009).

It is often assumed that temperature affects the molecular clock directly and identically in all clock cells, as opposed to light, which requires dedicated input pathways. However, in the adult, thermocycles phase the brain clock differently from all peripheral clocks, as judged from whole-tissue oscillations of a luciferase reporter enzyme (Glaser, 2005). Recent data suggest that subsets of clock neurons in the Drosophila adult brain may indeed be dedicated to temperature entrainment. In experiments combining LD and HC entrainment, all DN groups, as well as the less studied lateral posterior neurons (LPNs), seem to preferentially follow thermocycles, whereas the other LNs preferentially follow LD cycles (Miyasako, 2007). Although adult PDF⁺ LNs are able to entrain to thermocycles in the absence of any other functional clock, they do not seem to be required for (and actually slowed down) the temperature entrainment of activity rhythms, whereas the PDF-negative LPNs appear to play a prominent role in such conditions (Picot, 2009).

The current results indicate that a similar specialization toward light or temperature entrainment exists in the larval brain. The DN2s, which appear to be the most temperature-responsive clock neurons, are by themselves completely blind. Conversely, the four PDF-positive LNs, which may be the most light-sensitive clock neurons (with both CRY and the visual system as inputs), appear almost temperature blind, and depend on the DN2s for temperature entrainment. PER-negative DN2s do not allow PER oscillations in the larval LNs, suggesting that entrainment of the latter in HC cycles depends on clock function in the former. The hierarchy of clock neurons thus appears very different during entrainment of the clock network by one or the other Zeitgeber (Picot, 2009).

Effects of Mutation or Deletion

pdf-null animals were discovered among laboratory stocks as a fortuitous consequence of studying ectopic P element reporter gene expression. The pWF6-84 stock contains a dFMRF-lacZ fusion gene that produces ectopic reporter expression in a pattern that includes the LN_v neurons. pWF6-84 was mobilized and several lines were found that lacked all anti-PAP immunostaining and lacked beta-PDH immunostaining in most beta-PDH-positive cells. The lack of PAP immunostaining was heritable and specific for the PDF transmitter system. W33 and W15 lines were both positive when stained with other anti-neuropeptide antibodies (proFMRF). In W15 animals, beta-PDH signals were absent in LN_v, and in PDF-Tri and PDF-Ab neurons. However, signals were retained in neurons of the pars intercerebralis (PI) and protocerebrum (PL). PI neurons are present in both W33 and W15 flies. Neurons lacking beta-PDH signals in W15 correspond to those that express PDF mRNA and PDF in wild-type; this supports the hypothesis that PI and PL neurons do not express the Pdf gene product but instead express cross-reacting PDF related-antigen(s). The pdf gene from W15 animals was sequenced and a nonsense mutation was found at prepro-PDF residue 21, converting a Tyr to a stop codon. This mutation is referred to as pdf⁰¹. The conceptual pdf⁰¹ precursor is consistent with the immunostaining phenotype because it is truncated before epitope positions assayed by anti-beta-PDH, PDF, or PAP antibodies. With a PCR-based assay, the pdf⁰¹ mutation was found in each of the 22 derivative lines that displayed the phenotype. The same assay revealed that pdf⁰¹ is present in various laboratory stocks at a range of frequencies: some are homozygous for the allele. In a survey of 38 y w^67c23 adults, the stock into which the pWF6-84 DNA was injected, 9 flies homozygous and 20 heterozygous for pdf⁰¹ were found (Renn, 1999).

The lack of peptide expression could result from either the absence of PDF neurons or merely a lack of peptide transmitter expression in otherwise normal neurons. To determine the state of pdf neurons in animals containing the transmitter mutation, pdf-GAL4 was used to produce beta-galactosidase (beta-gal) in flies that contained a single pdf⁺ allele or that were hemizygous for pdf⁰¹. In all pdf⁰¹/deletion specimens, the number and morphology of LN_v, PDF-Tri, and PDF-Ab neurons were normal (Renn, 1999).

Over the course of 24 hr in light:dark (LD) cycles, wild-type flies are active at dawn, quieter at midday, then active again toward evening. One feature of flies entrained to LD cycling is the anticipation of transitions between lights-on and -off. Rhythmic behavior persists when wild-type flies proceed from LD into constant darkness. The locomotor activity rhythms of the pdf⁰¹ mutant were examined. Homozygous and hemizygous pdf⁰¹ flies are well entrained during LD cycles. However, pdf⁰¹ behavior in LD is not entirely normal. The evening activity peak is advanced by approximately 1 hr. Also, there is a lack of lights-on anticipation. Free-running behavior of pdf⁰¹ in constant darkness (DD) is severely abnormal and includes several features that distinguish this mutation from others that disrupt circadian behavior. By periodogram analysis, flies homozygous and hemizygous for pdf⁰¹ are much less rhythmic in DD than are pdf⁺ controls. Of these mutants, 50%-98% exhibited no detectable rhythmicity for the duration of 9 DD days. Actograms of pdf⁰¹ individuals suggest that most are rhythmic for 2 or 3 days in DD but later lose rhythmicity. Separate average activity histograms for DD days 1-2 and DD days 3-9 reveal the severity of the pdf⁰¹ phenotype. A higher proportion of mutant individuals are arrhythmic during DD days 3-9 than during DD days 1-9. The minority of pdf⁰¹ animals that maintain DD rhythmicity have free-running periods approximately 1 hr shorter than wild-type or pdf⁰¹/+ heterozygous flies (Renn, 1999).

Signal-to-noise analysis (SNR) was used to obtain measures that are linearly related to rhythm strength. SNR stems from Maximum Entropy Spectral Analysis (MESA) of the locomotor records: it provides single values for each fly's behavior and permits quantitation of weak rhythmicity, such as displayed by pdf⁰¹ flies. The average SNR for pdf⁺ flies depends on the background genotype and ranges from 1.7 for wild type to 1.0 for y w. Both stocks display normal proportions of rhythmic individuals and periods. The range of SNR values associated with the behavior of wild-type flies is very wide (0.4-4). In contrast, the distributions for the behaviorally arrhythmic per⁰¹ and disco mutants are skewed to the left, as expected. The distributions of per⁰¹ SNRs for DD days 1-9 and those for the DD days 3-9 are congruent. This reflects the fact that this period mutant is arrhythmic throughout the DD period. SNRs for pdf⁰¹ mutant animals are similar to those of per⁰¹, even when computed for the entire DD 1-9. Hence, the residual rhythms displayed by pdf⁰¹ animals in DD are very weak (Renn, 1999).

In Drosophila, the amidated neuropeptide Pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. It was asked whether other neuropeptides contribute to such auxiliary mechanisms. The gal4/UAS system was used to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restrict PHM expression to different numbers of peptidergic neurons. These mosaics display aberrant locomotor rhythms to degrees that parallel the apparent complexity of the spatial patterns. Certain PHM mosaics are less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms (Taghert, 2001).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Drosophila free-running rhythms require intercellular communication: Persistent molecular rhythm requires Pigment-dispersing factor

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN(v)s), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN(v)-specific driver, functional clocks were restricted to these neurons and it was shown that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, it was found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires Pigment-dispersing factor (PDF), an LN(v)-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN(v)s themselves. The brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, since it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN(v)s alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, it is proposed that LN(v)s, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms (Peng, 2003).

The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is required to maintain behavioral rhythms under constant conditions. To understand how PDF exerts its influence, time-series immunostainings were performed for the Period protein in normal and pdf mutant flies over 9 d of constant conditions. Without pdf, pacemaker neurons that normally express PDF maintained two markers of rhythms: that of Period nuclear translocation and its protein staining intensity. As a group, however, they displayed a gradual dispersion in their phasing of nuclear translocation. A separate group of non-PDF circadian pacemakers also maintained Period nuclear translocation rhythms without pdf but exhibit altered phase and amplitude of Period staining intensity. Therefore, pdf is not required to maintain circadian protein oscillations under constant conditions; however, it is required to coordinate the phase and amplitude of such rhythms among the diverse pacemakers. These observations begin to outline the hierarchy of circadian pacemaker circuitry in the Drosophila brain (Lin, 2004).